Antibiotic from Streptomyces clavulicerus

ABSTRACT

A new antibacterially active agent has been isolated from Streptomyces clavuligerus. This new compound which is designated clavulonic acid has the formula (I): ##STR1## In addition to being a broad spectrum antibiotic of medium potancy, clavulanic acid and its salts and esters have the ability to enhance the effectiveness of β lactam antibiotics against many β-lactamase producing bacteria.

CROSS-REFERENCE

This is a continuation of Ser. No. 078,292 filed Sept. 24, 1979, nowabandoned which is a division of Ser. No. 964,035 filed Nov. 27, 1978,now abandoned which is a continuation of Ser. No. 726,480 filed Sept.24, 1976 now abandoned which itself is a divisional of Ser. No. 569,007filed Apr. 17, 1975.

BACKGROUND OF THE INVENTION

a. Streptomyces clavuligerus has been described in detail by Higgens etal, Int.J.Systematic Bacteriology, 21, 326 (1971). This streptomycetewas of interest because it produced certain β-lactam antibiotics such aspenicillin N,7-(5-amine-5-carboxyvaleramido)-3-carbamoyloxymethyl-3-cephem-4-carboxylicacid and7-(5-amino-5-carboxyvaleramido)-3-carbamoyloxymethyl-7-methoxy-3-cephem-4-carboxylicacid. The streptomycete has been deposited in the Agricultural ResearchService Collection as NRRL 3585 and in the American Type CultureCollection as ATCC 27064. Streptomyces clavuligerus has also beenreferred to in U.S. Pat. No. 3,770,590 and also by Nagarajan et al,J.Amer.Chem.Soc., 93, 2308 (1971), Brannon et al, Antimicrob. AgentsChemother., 1, 237 (1972) and Antimicrob. Agents Chemother, 1, 247(1972) and Higgens et al, J.Antibiotics, 27, 298 (1974).

b. β-lactamases are enzymes which open the β-lactam ring of penicillinsand cephalosporins to give products which are devoid of antibacterialactivity. These enzymes are produced by many bacteria, notably speciesor strains of Escherichia, Klebsiella. Proteus, Pseudomonas,Enterobacter and Staphylococcus and are in many instances theexplanation for the resistance of certain strains of such organisms tosome penicillins and cephalosporins. The importance of β-lactamaseproduction may be understood when it is realised that a high proportionof clinically isolated organisms produce β-lactamases (see, for example,M. Wilson and I. A. Freeman, Bacteriological Proceedings, 80 (1969)where in a paper entitled `Penicillin Inactivation by Gram-negativeBacilli` they showed that 84% of the gram-negative organisms isolated inan American hospital produced β-lactamase). In many cases, somepenicillins or cephalosporins are ineffective in treating diseasesascribed to non β-lactamase-producing organisms because of the commonoccurrence of co-infection by a β-lactamase producer (see, for example,R. May et al; Brit. J.Dis.Chest., 66, 185 (1972)). Combination of aβ-lactamase inhibiting substance with a penicillin or cephalosporinmight be expected to protect the latter from degradation by bacterialβ-lactamase and thereby enhance their antibacterial activity againstmany infective organisms. This process of enhancement of theantibacterial activity is called synergism when the antibacterialactivity of the combination is well in excess of the simple addition ofthe activities of the two separate substances. The β-lactamaseinhibiting component of the mixture is referred to as a synergist andsuch substances are valuable for increasing the antibacterial activityof penicillins and cephalosporins against resistant organisms. It is oneof the objects of this invention to provide such synergists.

c. Examples of the use of certain β-lactamase resistant semi-syntheticpenicillins and cephalesporins as β-lactamase inhibitors and synergistsfor penicillins and cephalosporins have already been described in theliterature, see for example, Sutherland et al., Nature, 201, 868 (1964);Sabath et al., Nature, 204, 1066 (1964); O'Callaghan et al., Antimicrob.Agents and Chemotherapy, 1968, 67 (1969). However, none of these knownagents have a dramatic effect on the spectrum of the other antibioticpresent in the mixture.

d. Certain actinomycete cultures have been described as producingβ-lactamase inhibiting substances which act synergistically withpenicillins or cephalosporins, for example, those cultures disclosed inBritish Pat. No. 1,363,075 and those described by Hata et al, J.Antibiotics, 25, 473 (1972) and Umezawa et al, J. Antibiotics, 26, 51(1973). None of these β-lactamase inhibitors of actinemycetal originhave yet been found to be of use in the clinic. Particularly noteworthyfeatures which distinguish clavulanic acid from other β-lactamaseinhibitors of actinomycetal origin are its extractability into organicsolvents from culture filtrate at pH2, its high stability in human bloodand its broad spectrum of anti-bacterial and β-lactamase inhibitingactivity, its low molecular weight and its high R_(f) values on paperchromatography using a variety of solvent systems.

DESCRIPTION OF THE INVENTION

We have discovered that the aerobic cultivation of Streptomycesclavuligerus in conventional nutrient media at about 25°-30° C. underroughly neutral conditions produces a β-lactamase inhibitory substancewhich also possesses antibacterial activity. We have designated this newmaterial `clavulanic acid`.

Clavulanic acid has the following properties:

(a) It is a carboxylic acid.

(b) It forms a sodium salt which has a characteristic infra-red spectrumsubstantially as shown in the drawing.

(c) It is able to inhibit the growth of strains of Staphylococcusaureus.

(d) It is able to synergise the antibacterial effect of ampicillinagainst β-lactamase producing strains of Escherichia coli, Klebsiellaaerogenes and Staphylococcus aureus.

(e) It is able to synergise the antibacterial effect of cephaloridineagainst the β-lactamase producing strains of Proteus mirabilis andStaphylococcus aureus.

(f) It forms a methyl ester which has a molecular weight (by massspectroscopy) of 213.0635 which corresponds to the formula C₉ H₁₁ NO₅.

Thus clavulanic acid may be regarded as a monobasic carboxylic acid ofthe formula C₈ H₉ NO₅ which in the form of its sodium salt has acharacteristic infra-red absorption spectrum substantially as shown inthe drawing.

The compound produced by Streptomyces clavuligerus which has the aboveproperties has the formula (II): ##STR2##

The clavulanic acid may be named3-(β-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylicacid.

The stereochemistry at C₅ and C₂ of the clavulanic acid is the same asthat found in naturally occurring penicillins and cephalosporins so thatclavulanic acid may be represented by the structural formula (I):##STR3##

Thus a fuller chemical name for clavulanic acid isZ-(2R,5R)-3-(β-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylicacid.

The great usefulness of clavulanic acid may be readily appreciated whenit is realized that certain strains of Klebsiella aerogenes A, thegrowth of which is not inhibited by the presence of 125 μg/ml. ofampicillin, amoxycillin, carbenicillin or benzyl penicillin or by thepresence of 10 μg/ml. of clavulanic acid, are inhibited by the presenceof less than 12.5 μg/ml. of the previously mentioned penicillins when 5μg/ml. of clavulanic acid is also present. Similar results have beenobserved for combinations containing various esters of clavulanic acid.For example, strains of Klebsiella aerogenes A, the growth of which isnot inhibited by 125 μg/ml. of ampicillin or by 10 μg/ml of clavulanicacid methyl ester are inhibited by less than 12.5 μg/ml. of ampicillinin the presence of 5 μg/ml. of the clavulanic acid methyl ester. It hasalso been found that strains of Staphylococcus aureus Russell, thegrowth of which is not inhibited by the presence of 100 μg/ml. ofampicillin or by 5 μg/ml of clavulanic acid, are inhibited by thepresence of less than 10 μg/ml. of ampicillin in the presence of 1μg/ml. of clavulanic acid. In tests on female mice, it has been foundthat blood and tissue levels of clavulanic acid considerably in excessof 5 μg/ml. can readily be achieved by subcutaneous administration of100 mg/kg of the sodium salt of clavulanic acid and that useful levelsof clavulanic acid can be obtained after oral administration of 100mg/kg of the sodium salt of clavulanic acid.

Accordingly, the present invention provides clavulanic acid ashereinbefore described and its salts and esters.

Most suitably the salts of clavulanic acid will be pharmaceuticallyacceptable salts such as the sodium, potassium, calcium, magnesium,aluminium, ammonium and substituted ammonium salts such as thetrimethylammonium, benzathine, procaine and like salts conventionallyformed with penicillins or cephalosporins. Non-pharmaceuticallyacceptable salts of clavulanic acid are also included within the scopeof this invention as they, as well as the pharmaceutically acceptablesalts, are useful intermediates in the preparation of esters ofclavulanic acid, for example, the sodium lithium or silver salts ofclavulanic acid may be reacted with benzyl bromide to form the usefulbenzyl ester of clavulanic acid.

Salts of clavulanic acid tend to be more stable than the parent acid perse and thus form a favoured aspect of this invention. Particularlysuitable salts of clavulanic acid include the sodium and potassium saltswhich have the formula (III) and (IV) respectively: ##STR4## Crystallineforms of such salts may contain water of hydration.

Suitable esters of clavulanic acid include those notionally derived fromalcohols such as methanol, ethanol, prepanol, butanol,2,2,2-trichloroethanol, 2,2,2-trifluoroethanol, benzyl alcohol,p-nitrobenzyl alcohol, phenol, acetoxymethanol, pivaloyloxymethanol,2-dimethylaminoethanol and other conventional alcohols. Various estersof clavulanic acid are useful intermediates in certain processes for thepurification of clavulanic acid. Many clavulanic acid esters are usefulsynergistic compounds. The activity of such esters might be due tohydrolysis of the ester to the parent acid.

When used herein the term ester includes esters notionally derived froman alcohol or thiol of the formula ROH or RSH where R is an organicresidue. Suitable groups R include alkyl, alkenyl, alkynyl, aryl,arylalkyl or other similar groups any of which may be substituted ifdesired. In order not to increase the molecular weight to anunreasonable extent, groups R do not normally include more than 16carbon atoms, more suitably, not more than 12 carbon atoms and mostsuitably, not more than 8 carbon atoms.

Preferably, the group R is notionally derived from an alcohol ROH or(less favourably) a thiol RSH which is pharmaceutically acceptable.

Suitable substituents which may be included in the group R includehalogen atoms and lower alkoxyl, hydroxyl, lower acyloxyl, loweralkylamino, lower dialkylamino and like groups. The term `lower` meansthat the group contains up to 6 carbon atoms, and preferably up to 4carbon atoms. Thus, for example, R may be a methyl, ethyl, n-propyl,iso-propyl, straight or branched butyl, pentyl, heptyl, octyl, nonyl,decyl, undecyl, dodecyl, vinyl, allyl, butenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, cyclohexadienyl,methylcyclopentyl, methylcyclohexyl, benzyl, benzhydryl, phenylethyl,napthylmethyl, phenyl, naphthyl, propynyl, tolyl, 2-chloroethyl,2,2,2-trichloroethyl, 2,2,2-tri-fluoroethyl, acetylmethyl,benzoylmethyl, 2-methoxyethyl, 2-dimethylaminoethyl,2-diethylaminoethyl, 2-piperidinoethyl, 2-morpholinoethyl,3-dimethylaminopropyl, p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl,p-bromobenzyl, m-chlorobenzyl, 6-methoxynaphthyl-2-methyl,p-chlorophenyl, p-methoxyphenyl or any like group as well as thosegroups which are known from the penicillin or cephalosporin arts toproduce esters known to be readily hydrolysed in vivo to the parentantibiotic.

Readily hydrolysable esters include, but are not limited to, those ofthe formulae (V) and (VI): ##STR5## wherein A₁ is a hydrogen atom,alkyl, aryl or aralkyl group; A₂ is a hydrogen atom or methyl group; A₃is an alkyl, aryl or aralkyl group; X is oxygen or sulphur; Y is oxygenor sulphur and Z is a divalent organic group. Esters of the formulae (V)and (VI) which fairly readily release the clavulanic acid into the bloodstream after administration include those wherein A₁ is a hydrogen atom,A₂ is a hydrogen atom or a methyl group and A₃ is a methyl, ethyl,propyl, butyl, benzyl, or phenyl group and those wherein X is oxygen, Yis oxygen and Z is --CH₂ CH₂ --, --CH:CH--, ##STR6##

When used in conjunction with the preceding formula the term `alkyl`includes alkyl of up to six carbon atoms; the term `aryl` includesphenyl, naphthyl or phenyl substituted by an inert substituent such as afluorine or chlorine atom or a methyl or methoxyl group or the like;when used herein the term `aralkyl` means an alkyl group substituted byan aryl group.

Particularly suitable esters of the formulae (V) and (VI) include thoseof the formulae (VII) and (VIII): ##STR7## wherein A₄ is a hydrogen atomor a methyl group, A₅ is a methyl, t-butyl or phenyl group and A₆ is ahydrogen atom or a methoxyl group.

Many esters of clavulanic acid differ from analogous esters ofpenicillins or cephalosporins in that they show an enhanced tendency tohydrolyse to clavulanic acid under mild conditions. Thus, for example,simple alkyl esters such as the methyl ester slowly hydrolyse toclavulanic acid in water buffered to pH7. Esters which undergo somehydrolysis under mild conditions are included within the formula (IX):##STR8## wherein R¹ is a hydrocarbon group of 1∝9 carbon atomsoptionally subtituted by halogen, lower alkoxy, lower acyl, hydroxyl,lower acyloxy or optionally salted basic groups of the formula NR² R³wherein R² is a hydrogen atom or a lower alkyl group, R³ is a hydrogenatom or a lower alkyl group or is attached to R² so that NR² R³ is a 5-or 6-membered ring.

When used with reference to formula (IX) the term `lower` means that thegroup contains 1-4 carbon atoms.

Suitably groups R¹ include alkyl and aralkyl groups optionallysubstituted by halogen, methoxyl, hydroxyl or salted NR² R³ groupswherein R² is a methyl or ethyl group and R³ is a methyl or ethyl groupor is joined to R² so that NR² R³ is a pyrrolidine, piperidine ormorpholine group.

Most suitably alkyl groups R¹ are straight chain groups of up to 6carbon atoms optionally substituted by one methoxyl, hydroxyl, saltedNR² R³ group or one chlorine, bromine or iodine atom or by a CCl₃ or CF₃group.

The esters of clavulanic acid of particular usefulness as synergists arethose which hydrolyse in mammalian tissues, especially human blood, toyield clavulanic acid or a salt thereof because it is believed thatclavulanic acid and its salts tend to be somewhat more usefulsynergistic agents than the esters per se. Many of the esters of theformulae (V)-(IX) are useful for this purpose.

A further group of particularly suitable esters of this invention arethose useful intermediates which are readily converted to clavulanicacid or a salt thereof by chemical or biochemical techniques which areknown from the penicillin or cephalosporin arts to be sufficiently mildnot to degrade reactive acid-labile β-lactam rings.

Most suitably, the ester is one removable by hydrogenolysis.Conventional esters for such a process include benzyl, substitutedbenzyl, benzhydryl, substituted benzhydryl, trityl and the like. Thebenzyl ester has proved particularly useful for this purpose.

By and large, the nature of any substituent in the ester moiety isunimportant as long as it does not interfere with the hydrogenolysisreaction.

Since clavulanic acid and its salts are useful intermediates in thepreparation of the desirable antibacterially active esters of thisinvention, this invention also provides clavulanic acid and its saltswhen used as chemical intermediates.

As has been previously stated, clavulanic acid and its salts and estershave valuable therapeutic properties. Accordingly, in a further aspect,this invention provides a pharmaceutical composition which comprisesclavulanic acid or a salt or ester thereof together with apharmaceutically acceptable carrier.

The compositions of the invention include those in a form adapted fororal, topical or parenteral use and may be used for the treatment ofinfection in mammals including humans.

Suitable forms of the compositions of this invention include tablets,capsules, creams, syrups, suspensions, solutions, reconstitutablepowders and sterile forms suitable for injection or infusion. Suchcompositions may contain conventional pharmaceutically acceptablematerials such as diluents, binders, colours, flavours, preservatives,disintegrants and the like in accordance with conventionalpharmaceutical practice in the manner well understood by those skilledin the art of formulating antibiotics.

Injectable or infusable compositions of the clavulanic acid or its saltsare particularly suitable as high tissue levels of the compound ofclavulanic acid can occur after administration by injection or infusion.Thus, one preferred composition aspect of this invention comprisesclavulanic acid or a salt thereof in sterile form.

Unit dose compositions comprising clavulanic acid or a salt or esterthereof adapted for oral administration form a further preferredcomposition aspect of this invention.

Under certain conditions, the effectiveness of oral compositions ofclavulanic acid and its salts and esters can be improved if suchcompositions contain a buffering agent or an enteric coating agent suchthat the compounds of the invention do not have prolonged contact withhighly acidic gastric juice. Such buffered or enterically coatedcompositions may be prepared in accordance with conventionalpharmaceutical practice.

The clavulanic acid or its salt or ester may be present in thecomposition as sole therapeutic agent or it may be present together withother therapeutic agents such as a β-lactam antibiotic. Suitableβ-lactam antibiotics for inclusion in such synergistic compositionsinclude not only those known to be highly susceptible to β-lactamasesbut also those which have a good degree of intrinsic resistance toβ-lactamases. Thus, suitable β-lactam antibiotics for inclusion in thecompositions of this invention include benzylpenicillin,phenoxymethylpenicillin, carbenicillin, methicillin, propicillin,ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin,6-aminopenicillanic acid, 7-aminocephalosporanic acid,7-aminodesacetoxycephalosporanic acid, cephaloridine, cephalothin,cefazolin, cephalexin, cefoxitin, cephacetrile, cephamandole,cephapirin, cephradine, cephaloglycine and other well known penicillinsand cephalosporins or pro-drugs therefor such as hetacillin,metampicillin, the acetoxymethyl, pivaloyloxymethyl, or phthalidylesters of benzylpenicillin, ampicillin, amoxycillin or cephaloglycine orthe phenyl, tolyl or indanyl α-esters of carbenicillin or ticarcillin orthe like.

Naturally if the penicillin or cephalosporin present in the compositionis not suitable for oral administration then the composition will beadapted for parenteral administration.

When present in a pharmaceutical composition together with a β-lactamantibiotic, the ratio of clavulanic acid or its salt or ester present toβ-lactam antibiotic present may be from, for example, 20:1 to 1:12, moreusually 10:1 to 1:10.

The total quantity of antibacterial agents present in any unit dosageform will normally be between 50 and 1500 mg and will usually be between100 and 1000 mg.

Compositions of this invention may be used for the treatment ofinfections of inter alia, the respiratory tract, the urinary tract andsoft tissues in humans.

Compositions of this invention may also be used to treat infections ofdomestic animals such as mastitis in cattle.

Normally between 50 and 6000 mg of the compositions of the inventionwill be administered each day of treatment but more usually between 500and 3000 mg. of the composition of the invention will be administeredper day. However for the treatment of severe systemic infections orinfections of particularly intransigent organisms, higher doses may beused in accordance with clinical practice.

The exact form of the compositions of this invention will depend to someextent on the micro-organism which is being treated. For treatment ofmost infections the compositions of this invention are normally adaptedto produce a peak blood level of at least 0.1 μg/ml, more suitably atleast 0.25 μg/ml, and preferably at least 1 μg/ml of synergist, forexample, 2.5-5 μg/ml. of synergist.

The penicillin or cephalosporin in synergistic compositions of thisinvention will normally be present by up to or at approximately theamount conventionally used when that penicillin or cephalosporin is thesole therapeutic agent used in the treatment of infection.

Particularly favoured compositions of this invention will contain from150-1000 mg of amoxycillin, ampicillin or a pro-drug therefor and from50-500 mg of clavulanic acid or a salt or in-vivo hydrolysable esterthereof and more suitably from 200-500 mg of amoxycillin, ampicillin ora pro-drug therefor and from 50-250 mg of clavulanic acid or a salt orin-vivo hydrolysable ester thereof.

The materials present in such compositions may be hydrated if required.The weights of the antibiotics in such composition are expressed on thebasis of antibiotic theoretically available from the composition and noton the basis of the weight of pro drug.

When used herein the term "pro-drug" of an antibacterially active drugmeans any medicament which is known to be converted in the body to theantibacterially active drug per se.

This invention also provides a method of treating bacterial infection ina mammal which method comprises administering to the mammal anantibacterially effective amount of clavulanic acid or a salt or esterthereof.

Most suitably a pharmaceutically acceptable salt or in-vivo hydrolysableester of clavulanic acid is used.

This invention also provides a method of treating bacterial infection ina mammal, which method comprises administering to the mammal asynergistically effective amount of clavulanic acid or a salt or esterthereof and an antibacterially effective amount of a β-lactamantibiotic.

Most suitably a pharmaceutically acceptable salt or in-vivo hydrolysableester of clavulanic acid is used.

A further aspect of this invention provides a method of treatinginfections in humans caused by Klebsiella aeroginosa, which methodcomprises administering to an infected human a daily dose of (a) atleast 500 mg of ampicillin, amoxycillin or a pro-drug for ampicillin oramoxycillin, and (b) at least 100 mg of clavulanic acid or a salt orin-vivo hydrolysable ester thereof.

The penicillin and synergist may be administered in separatecompositions or in synergistic compositions containing both components.Normally the daily dose of the antibiotics will be administered individed form, for example, as 2 to 5 doses per day. Usually theantibiotics will be administered as 3 or 4 doses per day.

The penicillin used in this treatment may be anhydrous ampicillin,ampicillin trihydrate, sodium ampicillin, hetacillin, pivampicillinhydrochloride, talampicillin hydrochloride, amoxycillin trihydrate,sodium amoxycillin or the like. Each unit dose will usually contain from200-1000 mg of the penicillin, for example, 250 to 500 mg.

The synergist used in this treatment will generally be a salt or in-vivohydrolysable ester of clavulanic acid such as the sodium or potassiumsalt of clavulanic acid or the acetoxymethyl, pivaloyloxymethyl,phthalidyl or like ester of clavulanic acid. Each unit dose will usuallycontain from 50 to 500 mg of the synergist, for example, 100 to 250 mg.

A further aspect of this invention provides a method of treatinginfections in humans caused by Pseudomonas aeroginosa, which methodcomprises administering to an infected human a daily dose of at least 1g. of carbenicillin or ticarcillin or a pro-drug for carbenicillin orticarcillin and (b) at least 0.5 g. of clavulanic acid or a salt orin-vivo hydrolysable ester thereof.

The penicillin and synergist may be administered in separatecompositions or synergistic compositions containing both components.Normally, the daily dose of antibiotics will be administered in dividedform, for example, as 2 to 5 doses per day. Usually the antibiotics willbe administered as 3 or 4 doses per day. For systemic or severalinfections the compositions will be adapted for administration byinjection or infusion. For infections of the urinary tract thecompositions may be adapted for administration orally or by injection orinfusion.

The penicillin used in this treatment may be carbenicillin,carbenicillin α-phenyl ester, carbenicillin α-5-indanyl ester,ticarcillin, ticarcillin α-tolyl ester, ticarcillin α-phenyl ester andlike, and will usually be in the form of a salt such as a sodium salt.Each unit dose will usually contain from 400 to 4000 mg of thepenicillin, for example, 500 to 1000 mg.

The synergist used in this treatment will suitably be the sodium orpotassium salt of clavulanic acid or an in-vivo hydrolysable esterthereof, such as the acetoxymethyl, pivaloyloxymethyl or phthalidylester of clavulanic acid. Each unit dose will usually contain from 200to 1000 mg of the synergist, for example, 250 to 750 mg.

A further aspect of this invention provides a method of treatinginfections in the respiratory tract of humans, which method comprisesadministering to an infected human a daily dose of (a) at least 500 mgof amoxycillin or ampicillin or a pro-drug for ampicillin oramoxycillin, and (b) at least 100 mg of clavulanic acid or a salt orin-vivo hydrolysable ester thereof.

Especially suitable doses and methods of administration are similar tothose described for the treatment of infections due to Klebsiellaaeroginosa.

A further aspect of this invention provides a method of treatinginfections in the urinary tract in humans which method comprisesadministering to an infected human a daily dose of (a) at least 500 mgof ampicillin, amoxycillin, carbenicillin, ticarcillin, cephalothin,cephaloridine, cephaloglycine, cephalexin, cefazolin, cephapirin orcephradine or a pro-drug for such medicaments and (b) at least 100 mg ofclavulanic acid or a salt or in-vivo hydrolysable ester thereof.

The medicaments may be administered in manner analogous to thatdescribed above for the treatment of infections due to Klebsiellaaeroginosa.

In a process aspect, the present invention provides a process for thepreparation of clavulanic acid and salts and esters thereof whichprocess comprises cultivating a strain of Streptomyces clavuligerus andrecovering clavulanic acid or a salt thereof from the culture medium andthereafter if desired, forming the free acid or a salt or ester bymethods known per se.

Preferably, Streptomyces clavuligerus ATCC 27064 or a high yieldingmutant thereof is used in the process of this invention.

When used herein, the term `cultivation` means the deliberate aerobicgrowth of a clavulanic acid producing organism in the presence ofassimilable sources of carbon, nitrogen and mineral salts. Such aerobicgrowth may take place in a solid or semi-solid nutritive medium, or in aliquid medium in which the nutrients are dissolved or suspended. Thecultivation may take place on an aerobic surface or by submergedculture. The nutritive medium may be composed of complex nutrients ormay be chemically defined. In our hands we have found media containingcomplex nutrients such as yeast extract, soya bean flour and the like tobe particularly suitable.

The nutrient media which may be used for the cultivation of Streptomycesclavuligerus may contain, in the range 0.1-10% a complex organicnitrogen source such as yeast extract, corn steep liquor, vegetableprotein, seed protein, hydrolysates of such proteins, milk proteinhydrolysates, fish and meat extracts and hydrolysates such as peptones.Alternatively chemically defined sources of nitrogen may be used such asurea, amides, single or mixtures of common amino acids such as valine,asparagine, glutamic acid, proline and phenylalanine. Carbohydrate(0.1-5%) may be included in the nutrient media but glucose in certainmedia is undesirable having a depressing effect on the yield of thedesired clavulanic acid. Starch or starch hydrolysates such as dextrin,sucrose, lactose or other sugars or glycerol or glycerol esters may alsobe used. The source of carbon may also be derived from vegetable oils oranimal fats. Carboxylic acids and their salts can be included as asource of carbon for growth and production of β-lactamase inhibitors. Aparticularly suitable low cost medium is one containing soya bean flour(Arkasoy) plus dried malt distillers solubles (Scotasol) plus dextrin.

The addition of antifoam agents such as Pluronic L81 may be necessary tocontrol foaming of certain media in fermenters.

Mineral salts such as NaCl, KCl, MgCl₂, ZnCl₂, FeCl₃, Na₂ SO₄, FeSO₄,MgSO₄ and Na⁺ or K⁺ salts of phosphoric acid may be added to the mediadescribed above particularly if chemically defined; CaCO₃ may be addedas a source of Ca⁺⁺ ions or for its buffering action. Salts of traceelements such as nickel, cobalt or manganese may also be included.Vitamins may be added if desired.

When used herein the term `mutant` includes any mutant strain whicharises spontaneously or through the effect of an external agent whetherthat agent is applied deliberately or otherwise. Suitable methods ofproducing mutant strains include those outlined by H. I. Adler inTechniques for the Development of Micro-Organisms in `Radiation andRadioisotopes for Industrial Micro-Organisms`, Proceedings of aSymposium, Vienna, 1973, page 241, International Atomic Energy Authorityand these include:

i. Ionising radiation (such as X- and γ-rays), uv light, uv light plus aphotosensitizing agent (such as 8-methoxypsoralen), nitrous acid,hydroxylamine, pyrimidine base analogues (such as 5-bromouracil),acridines, alkylating agents (such as mustard gas, ethyl-methanesulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and,

ii. Genetic techniques such as recombination, transformation,transduction, lysogenisation, lysogenic conversion and selectivetechniques for spontaneous mutants.

Cultivation of Streptomyces clavuligerus normally takes place in thetemperature range 15°-50° C., usually 20°-35° C. and preferably, 25°-30°C. and at a pH of between 5 and 8.5, preferably between 6 and 7.5.

The Streptomyces clavuligerus may be cultivated in the above media inglass conical flasks aerated by shaking on a rotary shaker or in baffledstainless steel fermenters stirred with vaned disc impellers and aeratedwith a sparger. The fermentation may also be carried out in a continuousfashion.

The starting pH of the fermentation is typically 7.0 and maximum yieldof clavulanic acid is obtained in 2-10 days at 20°-35° C. In a stirredstainless steel fermenter using the Arkasoy/Scotasol/Dextrin mediumreferred to above the preferred temperature is 26° C. and peak yieldsclavulanic are obtained with 5 days.

Clavulanic acid may be extracted from culture filtrate by a variety ofmethods. Solvent extraction from cold culture filtrate adjusted to acidpH values and methods based on the anionic nature of the metabolite suchas the use of anion exchange resins have been found to be particularlyuseful. The cells of Streptomyces clavuligerus are normally firstremoved from the fermentation by filtration or centrifugation beforesuch extraction procedures are commenced.

In the solvent extraction process, the culture filtrate is chilled andthe pH lowered into the region of pH 2-3 by the addition of acid whilethoroughly mixing with a water immiscible organic solvent such asn-butylacetate, methylisobutylketone, n-butanol or ethylacetate. Theacid used to lower the pH of the medium is normally a mineral acid suchas hydrochloric, sulphuric, nitric, phosphoric or the like acid.n-Butanol is a particularly suitable solvent for use in separation ofthe phases by centrifugation, the β-lactamase inhibiting metabolite isback extracted from the solvent phase into aqueous sodium bicarbonate orpotassium hydrogen phosphate buffer, CaCO₃ suspension or water whilemaintaining the pH at approximate neutrality, for example, at pH 7.0.This aqueous extract after separation of phases may be concentratedunder reduced pressure and freeze dried to give a crude preparation of asalt of clavulanic acid. This preparation is stable when stored as a drysolid at -20° C.

In the anion exchange resin process, the clarified culture filtrate atan approximately neutral or slightly acid pH, for example pH 6-7, ispercolated down a column of weak or strong base anion exchange resinsuch as Amberlite IR4B or Zerolite FFIF respectively until the resin issaturated and the β-lactamase inhibiting material emerges from thebottom. The column is then washed with water and eluted with aqueoussodium chloride. The β-lactamase inhibiting fractions are collected,bulked, desalted and freeze dried to yield a crude solid salt ofclavulanic acid. Amberlite IR 4B is an example of a weakly basic anionexchange resin with polyamine active groups and cross linkedpolystyrene-divinyl-benzene matrix. Zerolite FFIP is a strongly basicanion exchange resin with quaternary ammonium active groups and a crosslinked polyvinyldivinylbenzene matrix. Resins similar to Zerolite FFIPinclude Isopor FFIP and DeAcidite FFIP SRA.64. These resins weresupplied by BDH Chemicals Ltd., Poole, Dorset, U.K.

An alternative form of the extraction process is to contact the culturefiltrate (usually at approximately neutral pH) containing a salt ofclavulanic acid, with an organic phase in which is dissolved a waterinsoluble amine. Suitable organic solvents include such conventionalwater immiscible polar solvents as methylisobutylketone,trichloroethylene and the like. Suitable amines include secondary ortertiary amines in which one of the substituent groups is a long chainaliphatic group, for example, of 12-16 carbon atoms and the other is atertiaryalkyl group so that the molecule is lipophilic. In our handsAmberlite LA2 has proved a successful amine. Normally the amine is usedas its acid addition salt. After this extraction process the clavulanicacid is present in the organic phase as the amine salt. The organicphase is then separated from the culture filtrate. The clavulanic acidmay be back extracted into an aqueous phase by back extraction with asalt solution, preferably a concentrated solution of sodium chloride,sodium nitrate or the like. The crude salt of clavulanic acid may thenbe obtained by freeze drying or the like.

Other primary methods of isolation which may be used includeconventional methods such as adsorption onto carbon, precipitation,salting out and molecular filtration but these methods are not usuallyas successful as the above described methods which are preferred.

Further purification of the crude solids obtained by methods describedabove may be obtained by a variety of methods but ion exchange columnchromatography is particularly suitable especially when using Isopor,DeAcidite FFIP SRA64 or DEAE cellulose. The DeAcidite column may begradient eluted with aqueous solution of a salt such as sodium chloride(0-0.5M). The column of DEAE cellulose in 0.01M phosphate buffer at pH7may be eluted with a salt solution, normally a NaCl solution (0-0.2MNaCl in 0.01M phosphate buffer pH7). Active fractions may be detected bytheir β-lactamase inhibitory activity and their antibacterial activityagainst Klebsiella aerogenes in an agar diffusion assay. The fractionscontaining the bulk of this activity are then combined and concentratedto a small volume under vacuum. This crude preparation of the clavulanicacid salt is desalted by percolating down a column of Bio Gel P2.

(Bio Gel P2 is an example of a highly lipophilic resin onto whichorganic materials may be adsorbed but which does not retain inorganicsalts. Bio Gel P2 is a polyacrylamide gel supplied by Bio Rad, 32nd andGriffen Avenue, Richmond, Calif. 94804, USA). The active desaltedmaterial is then concentrated, mixed with ethanol and furtherchromatographed on a cellulose column using butanol/ethanol/water 4/1/5v/v top phase as solvent.

Fractions containing material which inhibit Escherichia coli β-lactamaseare bulked, evaporated to dryness under vacuum, redissolved in water andfreeze dried to give a salt of clavulanic acid as a white solid.

The methods we have found most useful in detecting clavulanic acid inculture filtrates are paper chromatography and a bioautographicdetection system. Clavulanic acid may be assayed by making use of itsβ-lactamase inhibiting activity. Thin layer chromatography may be usedto detect clavulanic acid in solid preparations. These detection andassay procedures are described hereinafter.

A variation of the process for the preparation of a pure form ofclavulanic acid or its salts comprises isolating an impure form ofclavulanic acid or salt thereof, forming an ester of clavulanic acid inconventional manner, purifying the ester and thereafter regeneratingclavulanic acid or a salt thereof from the ester.

The impure clavulanic acid or its salts used in this process willnormally contain at least 1% by weight of the antibiotic.

Suitable esters for use in this process include those which may becleaved by hydrogenolysis, enzymatic methods or by hydrolysis under verymild conditions.

One suitable group of esters used in this process is that of the formula(X): ##STR9## wherein A₇ is a hydrogen atom or an optionally substitutedphenyl group and A₈ is an optionally substituted phenyl group.

Most suitably A₇ is a hydrogen atom or a phenyl, tolyl, chlorophenyl ormethoxyphenyl group and A₈ is a phenyl, tolyl, chlorophenyl ormethoxyphenyl group.

Preferably A₇ is a hydrogen atom and A₈ is a phenyl group.

The esters of formula (X, may be cleaved by hydrogenolysis to yieldclavulanic acid or a salt thereof.

Other groups of esters which may be used in this process include thoseof formulae (V) and (VI) as hereinbefore described. Such esters may beconverted to salts of clavulanic acid by mild alkaline hydrolysis, forexample, at pH 7.5.

The impure form of clavulanic acid or salt thereof which is to bepurified in this process may be in the form of a solid or solution whichwill usually also contain considerable quantities of organic orinorganic impurities.

The clavulanic acid or salt thereof may be converted into an ester bythe esterification reactions referred to hereinafter. The preferredmethod of forming the required ester of clavulanic acid is by thereaction of a salt of clavulanic acid with an esterifying agent such asa reactive halide, sulphonate ester or the like as hereinafterdescribed. Such reactions are frequently carried out in an organicsolvent of high dielectric constant such as dimethylformamide,dimethylformamide/acetone, dimethylsulphoxide, N-methylacetamide,hexamethylphosphoramide and the like.

If desired the salt of clavulanic acid may be dissolved in the solventin conventional manner or it may be bound to a polymeric support.Suitable supports for use in this process include strong base anionexchange resins, especially those possessing a macroreticular naturewhich permits the use of non-aqueous solvent systems. We have foundAmerblyst A26 to be suitable for this purpose. The clavulanic salt maybe adsorbed onto the resin from the culture filtrate and the resin thensuspended in dimethylformamide containing sodium iodide or alternativelyeluted columnwise with a solution of sodium iodide in dimethylformamideor in a mixture of dimethylformamide and acetone.

Once formed, the impure ester of clavulanic acid is normally purifiedchromatographically. In such procedures the ester is normally dissolvedin an organic solvent such as ethylacetate, methylene chloride,chloroform, cyclohexane or similar solvents. The solid phase used in thechromatographic process is normally an inert material such as silica gelor chromatographically similar materials.

The fractions emerging from the column may be tested for the presence ofthe clavulanic acid by making use of its synergistic properties. Activefractions are normally combined and the organic solvent evaporated offunder reduced pressure.

The ester resulting from this process is generally of acceptable purity,but the material may be rechromatographed if desired.

This purified ester of clavulanic acid may be converted to clavulanicacid or a salt thereof by the before mentioned methods.

A particularly suitable method of obtaining clavulanic acid or its saltis by hydrogenation of a compound of the formula (X) as hereinbeforedescribed. Such reactions normally take place in the presence of atransition metal catalyst using low or medium pressures of hydrogen. Thereaction may be carried out at high, ambient or depressed temperatures,for example at 0°-100° C. Particularly suitable reaction conditions forsuch hydrogenations will use a slightly superatmospheric pressure ofhydrogen at an approximately ambient (12°-20° C.) temperature. Thereaction may be carried out in conventional solvents such as loweralkanols, for example, ethanol. We have found that a particularlysuitable catalyst is palladium on charcoal.

If the hydrogenation is carried out in the presence of a base then asalt of clavulanic acid is produced, for example, the sodium orpotassium salts result if the reaction is carried out in the presence ofsodium or potassium hydrogen carbonate.

The clavulanic acid or salt thereof resulting from such reactions isgenerally of good purity.

Esters or clavulanic acid may be prepared by the esterification ofclavulanic acid or a salt thereof by conventional methods.

Suitable methods of ester formation include

(a) reaction of a salt of the acid of clavulanic acid with a compound ofthe formula Q--R where Q is a readily displaceable group and R is anorganic group;

(b) the reaction of clavulanic acid with a diazoalkane and (c) thereaction of clavulanic acid with an alcohol ROH in the presence of acondensation promoting agent such as carbodiimide or the like.

Suitable salts of clavulanic acid which may be reacted with compoundsR--Q include alkali metal salts such as the sodium or potassium salts orother conventional salts such as the silver salt

Suitable groups Q include those atoms or groups known to be displaceableby carboxylate anions and include chlorine, bromine and iodine atoms,sulphonic acid esters such as the O.SO₂ CH₃ or O.SO₂ C₆ H₄ CH₃ groups,active ester groups such as the O.CO.H or O.CO.CF₃ group and otherconventional groups displaceable by nucleophiles.

The preceding reaction is normally carried out in an organic solvent ofrelatively high dielectric constant such as dimethylformamide, acetone,dioxane, tetrahydrofuran or the like and at a non-extreme temperaturesuch as -5° C. to 100° C., more usually +5° C. to 30° C., for example atambient temperature.

The reaction of clavulanic acid with a diazocompound is a mild method ofmaking alkyl, aralkyl or similar esters. The diazotization reaction maybe performed under conventional reaction conditions, for example at anon-extreme temperature and in a conventional solvent. Such reactionsare normally carried out at between about -5° C. and 100° C., moreusually from 5° C. to 30° C., for example at ambient temperature.Suitable solvents for this reaction include lower alkanols such asmethanol and ethanol and solvents such as tetrahydrofuran, dioxane andthe like. Ethanol has proved a particularly useful solvent for thisreaction.

The reaction of clavulanic acid with an alcohol or thiol in the presenceof a condensation promoting agent will normally take place in an inertorganic solvent of relatively high dielectric constant such asacetonitrile. This reaction is usually carried out at an ambient ordepressed temperature, for example at -10° C. to +22° C., more usually-5° C. to +18° C., for example initially at 0° C. and thereaftergradually warming to about 15° C. The condensation promoting agent usedis normally one which removes water from the reaction mixture. Suitableagents include carbodiimides; carbodiimidazoles or equivalent reagents.Dicyclohexylcarbodiimide has proved to be a particularly suitablecondensation promoting agent for use in this process. In order tominimise self condensation of the clavulanic acid, this reaction isusually carried out in the presence of a considerable excess of thealcohol or thiol.

Other suitable methods of ester formation include (d) removal of theelements of carbon dioxide from a compound of the formula (XI) ##STR10##wherein R⁴ is an inert organic group; and (e) reaction of a compound ofthe formula (XI) with an alcohol ROH (or less favourably with a thiolRSH).

The elements of carbon dioxide may be removed from the compound offormula (XI) spontaneously during its preparation or alternatively byheating the compound of the formula (XI) in an inert solvent. Suitableinert solvents include ether solvents such as diethylether,tetrahydrofuran, dioxane and the like. In many cases the compound of theformula (XI) decomposes spontaneously even at a depressed temperature,for example, at -5° C., to yield an ester of the formula ##STR11##wherein R⁴ is an inert group within the definition of R.

When the compound of the formula (XI) is to be reacted with an alcohol(or less favourably with a thiol) then this reaction is normally carriedout in an inert solvent such as an ether solvent in the presence of anexcess of the alcohol (or thiol) in order to prevent self-condensationof the clavulanic acid derivative.

Such methods of esterification are not in general as useful as thoseinvolving reaction of a salt of clavulanic acid with R--Q ashereinbefore described.

The compound of the formula (XI) may be prepared by the reaction of asalt of clavulanic acid with Cl.CO.O.R⁴ or the chemical equivalentthereof. Normally this reaction is carried out at a depressedtemperature, for example, at a temperature not greater than 5° C., andin an inert solvent, for example diethylether, tetrahydrofuran, dioxaneand the like. Most suitably the salt of clavulanic acid used in thisreaction is a lipophilic salt so that it will dissolve in the solventalthough if desired the less favourable sodium salt may be employed bysuspending it in the reaction medium.

DESCRIPTION 1 ASSAY SUITABLE FOR DETECTION OF CLAVULANIC ACID Principleof the Assay

Solutions containing clavulanic acid (culture filtrate, samples fromisolation procedure and the like) are incubated for 15 minutes with aβ-lactamase preparation in 0.05M phosphate buffer at pH 7 and 37° C.During this time, enzyme inhibition or inactivation occurs. Substrate(benzylpenicillin) is then added and incubation continued for 30 minutesat 37° C. The amount of enzymic degradation of the substrate topenicilloic acid is determined by the hydroxylamine assay forpenicillin. The amount of β-lactamase used is such as to give 75%hydrolysis of the benzylpenicillin in 30 minutes at 37° C.

The extent of hydrolysis is a reflection of the amount of enzymeremaining uninhibited. The results are expressed as percent inhibitionof the enzyme activity by a given dilution of the clavulanicacid-containing solution (e.g. culture filtrate) or the concentration ofclavulanic acid (μg/ml) giving 50% inhibition of the enzyme under theabove stated conditions (I₅₀).

β-lactamase Enzyme

The β-lactamase produced by Escherichia coli JT4 is used as an enzyme.This culture is an ampicillin resistant strain and owes its resistanceto the production of an R-factor controlled β-lactamase. Other similarR-factor controlled β-lactamases may be used if desired.

The culture maintained on nutrient agar slopes, is inoculated into 400ml. of sterile Tryptone medium contained in a 2 liter conical flask.This medium has the following composition Tryptone (Oxoid) 32 g/l, yeastextract (Oxoid) 20 g/l, NaCL 5 g/l and CaCl₂ 6H₂ O 2.2 g/l. The final pHwas adjusted to 7.4 with dilute NaOH. The flask is shaken at 25° C. for20 hours on a rotary shaker at 240 r.p.m.

The bacterial cells are collected by centrifugation, washed with 0.05Mphosphate buffer pH 7 (resuspended and centrifuged) and resuspended inwater to give cell concentration 25 times that in the cultivationmedium. This cell suspension was then disrupted in an MSE ultrasonicdisintegrator at 4° C. The cell debris was removed by centrifugation andaliquots of the supernatant stored deep frozen. For use in the assayprocedure, the supernatant is diluted in 0.005M phosphate buffer untilit gives about 75% hydrolysis of a 1 mg/ml. solution of benzylpenicillinin 30 minutes at 37° C.

Assay Procedure

Suitable dilutions of the inhibitor preparation and β-lactamase solutionare mixed and incubated at 37° C. for 15 minutes (Test). A control withbuffer in place of inhibitor preparation is also incubated.Benzylpenicillin solution (substrate) is then added to test and controlmixtures, incubation continued for a further 30 minutes at 37° C. Theresidual benzylpenicillin in each mixture is then estimated using thehydroxylamine assay as described by Batchelor et al, Proc. Roy. Soc., B154, 498 (1961). 6 ml of hydroxylamine reagent are added to all tests,controls and blanks and are allowed to react for 10 minutes at roomtemperature prior to the addition of 2 ml of ferric ammonium sulphatereagent. The absorption of the final solutions is measured in an E.E.L.Colorimeter of a Spectrophotometer at 490 nm against the reagent blank.The composition of the reactions, tests and blanks prior to thehydroxylamine assay are as follows:

    ______________________________________                                        Components            Benzyl-                                                 (all dissolved in or  penicillin      Reagent                                 diluted with 0.005 M  Blank    Control                                                                              Blank                                   pH 7 phosphate buffer)                                                                       Test   ml.      ml.    ml.                                     ______________________________________                                        Escherichia coli                                                                             1.9    0.0      1.9    1.9                                     β-lactamase solution                                                     Inhibitor solution                                                                           0.1    0.0      0.0    0.0                                     Benzylpenicillin                                                                             0.5    0.5      0.5    0.0                                     5 mg/ml.                                                                      0.005 M pH 7 phosphate                                                                       0.0    2.0      0.1    0.6                                     buffer                                                                        ______________________________________                                    

Calculation of Results

The percentage inhibition of the β-lactamase is calculated as follows

Absorption of benzylpenicillin blank minus absorption of control(uninhibited reaction)=x

Absorption of test (inhibited reaction) minus absorption of control(uninhibited reaction)=y

    % inhibition=(y/x)×100

To obtain the I₅₀ value, the inhibitor preparation is diluted until 50%inhibition of the β-lactamase inactivation of benzylpenicillin isobtained in the above procedure.

DESCRIPTION 2 PAPER CHROMATOGRAPHIC DETECTION OF CLAVULANIC ACID

Culture filtrate and a reference solution of clavulanic acid (250 μg/mlpartially purified preparation), are spotted (20 μl/origin) onto WhatmanNo. 1 paper strips 1 cm. wide. The chromatograms are run by descendingchromatography for 16 hours at 5° C. using n-butanol/isopropanol/water,7/7/6 v/v as solvent. The strips are dried at 40° C. and laid on agarplates containing 6 μg/ml benzylpenicillin and seeded with a β-lactamaseproducing strain of Klebsiella aerogenes (synergism system). The platesare incubated overnight at 30° C. and clavulanic acid revealed as a zoneof inhibited growth. The R_(f) value of the zone was 0.46. The 6 μg/mlbenzylpenicillin alone is below the concentration required to kill theKlebsiella aerogenes but in the presence of a β-lactamase inhibitor,this concentration becomes toxic, that is to say there is synergism.

Use of the above synergism system enables clavulanic acid to be detectedat concentrations below those at which it shows antibacterial activity.

DESCRIPTION 3 THIN LAYER CHROMATOGRAPHIC DETECTION OF CLAVULANIC ACIDSODIUM SALT

Solutions of clavulanic acid sodium salt preparations are spotted (5 μlof 1 mg/ml) onto glass plates coated with a 0.25 mm layer of silica gel(F254) as supplied by E. Merck, Darmstadt, Germany. The chromatogramsare run at 22° C. using the top phase of the mixturen-butanol/ethanol/water 4/1/5 v/v. The chromatogram plates are dried at40° C. and clavulanic acid sodium salt located by bioautography on agarplates containing 6 μg/ml. benzylpenicillin and seeded with Klebsiellaaerogenes (synergism system--see section on paper chromatography above).The agar surface is covered by a fine filter cloth before laying the TLCplate onto it. After allowing 14-30 minutes for wetting and diffusion,the TLC plate is lifted off with the aid of the filter cloth and theagar plate incubated overnight at 30° C. to reveal the zones ofinhibited growth. The R_(f) value of clavulanic acid sodium salt in theabove solvent is approximately 0.37. Two spray reagents, Ehrlich andtriphenyltetrazolium chloride are also used to reveal the clavulanicacid sodium salt zone. The former reagent consists of 300 mg ofp-dimethylaminobenzaldehyde dissolved in 9 ml. of ethyl alcohol, 54 ml.of n-butanol and 9 ml of concentrated HCl. On heating the sprayed TLCplate at 120° C. for 1-2 minutes, clavulanic acid sodium salt appears asa pink spot. The triphenyltetrazolium chloride reagent consists of amixture of 1 volume of a 4% solution of this compound in methanol with 1volume of methanolic sodium hydroxide. After spraying, the TLC platesare heated at 80° C. Clavulanic acid sodium salt appears as a red spoton a white background.

EXAMPLE 1 Cultivation of Streptomyces clavuligerus

Streptomyces clavuligerus was cultivated at 26° C. on agar slopescontaining 1% Yeatex (yeast extract), 1% glucose and 2% Oxoid agar No.3, pH 6.8. A sterile loop was used to transfer mycelium and spores fromthe slope into 100 ml of a liquid medium in a 500 ml Ehrlenmyer flask.The liquid medium had the following composition:

Oxoid Malt Extract--10 g/l

Oxoid Bacteriological Peptone--10 g/l

Glycerol--20 g/l

Tap water--1 liter

The medium was adjusted to pH 7.0 with sodium hydroxide solution and 100ml. volumes dispensed into flasks which were closed with foam plugsprior to autoclaving at 15 lb/sq.in. for 20 minutes. An inoculated seedflask was shaken for 3 days at 26° C. on a rotary shaker with 2 inchthrow and a speed of 240 r.p.m. Production stage flasks containing theliquid medium described above were inoculated with 5% vegetativeinoculum and grown under the same conditions as the seed flask. Samplesof culture filtrate were assayed for inhibitor action against theβ-lactamase of Escherichia coli JT4. Optimum activity was obtained after3 days. The results are shown in Table 1. A zone of clavulanic acid atR_(f) 0.46 was seen when the culture filtrate was examined by the paperchromatographic method previously described. The increase in size of thezone paralleled the increase in the β-lactamase inhibitor assay.

Streptomyces clavuligerus was also cultivated in 2 liter shaken flaskscontaining 400 mls. of medium (Production stage) using the same mediumand cultural conditions as described earlier in this Example. In theselarger vessels, growth of the organism was slower and optimumβ-lactamase inhibitory activity was achieved 7-9 days after inoculationwith the vegetative seed. The results are also shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        β-Lactamase Inhibiting Activity of Streptomyces clavuligerus             Grown in 500 ml. and 2000 ml. Flasks                                                    % Inhibition of Escherichia coli                                    Fermentation                                                                            β-lactamase at a final dilution of                             Time      1/2500 of culture filtrate                                          (Days)    500 ml. Shaken Flask                                                                         2000 ml. Shaken Flask                                ______________________________________                                        1         15             --                                                   2         30             --                                                   3         55             --                                                   4         50             10                                                   5         51             21                                                   6         57             36                                                   7         --             51                                                   8         --             53                                                   9         --             50                                                   ______________________________________                                    

EXAMPLE 2 Cultivation of Streptomyces clavuligerus

A seed flask prepared as in Example 1 was used to inoculate 500 ml.conical flasks containing 100 ml. aliquots of the following medium indeionised water:

Soluble Starch--2% w/v

Glycerol--0.3% w/v

Scotasol--0.1% w/v

Arkasoy--1% w/v

FeSO₄.7H₂ O--0.01% w/v

The medium was sterilized by autoclaving at 15 p.s.i. for 20 minutes andinoculated by the addition of the 5% vegetative seed stage. The flaskswere shaken at 26° C. on a rotary shaker as in Example 1. Optimum titreof clavulanic acid was achieved between 3-5 days. A dilution of 1/2500of the culture filtrate gave 60% inhibition in the β-lactamaseinhibition assay. A zone of clavulanic acid was seen at R_(f) 0.46 whenusing the paper chromatographic (bioautographic) method previouslydescribed. This zone increased in size in parallel with the increase ofthe activity in the β-lactamase inhibitor assay.

[Soluble starch supplied by British Drug Houses, Ltd., Poole, U.K.;

Scotasol is dried distillers solubles supplied by Thomas Borthwich Ltd.,60 Wellington Street, Glasgow, UK;

Arkasoy is soya bean flour supplied by British Arkady Co., Old Trafford,Manchester, UK].

EXAMPLE 3 Cultivation of Streptomyces clavuligerus

A seed flask as produced in Example 1 was used to inoculate 500 ml.conical flasks containing 100 ml aliquots of the following mediumprepared in deionised water and sterilised as previously described. Theinoculum level was 5%.

Dextrin--2% w/v

Arkasoy--1% w/v

Scotasol--0.1% w/v

FeSO₄ 7H₂ O--0.01% w/v

The inoculated flasks were shaken at 26° C. Optimum β-lactamaseinhibitory activity was achieved between 3-5 days. The activity wassimilar to that achieved in Example 2.

[Dextrin is supplied by C P C (UK) Ltd., Trafford Park, Manchester, UK]

EXAMPLE 4 Cultivation of Streptomyces clavuligerus

The seed stage as described in Example 1 was used to inoculate 500 ml.conical flasks containing the following medium prepared in deionisedwater.

Dextrose--1% w/v

Soyabean Meal--1% w/v

Scotasol--0.05% w/v

CaCO₃ --1% w/v

These flasks were treated exactly as in previous Examples and culturedunder identical conditions. β-lactamase inhibitory activity was producedbetween 3-5 days. Culture filtrate at a final dilution of 1/2500 gave35-45% inhibition in the β-lactamase inhibition assay.

EXAMPLE 5 Cultivation of Streptomyces clavuligerus

β-lactamase inhibitory activity attributable to clavulanic acid wasproduced using the following medium with identical seed stage andcultivation conditions to Example 1.

Glycerol--2% w/v

Soyabean Meal--1.5% w/v

Mg SO₄ --0.1% w/v

K₂ HPC₄ --0.1% w/v

Medium prepared in deionised water

β-lactamase inhibitory activity reached a maximum level between 3-5 daysand was of a similar order to that produced in Example 4.

EXAMPLE 6 Cultivation of Streptomyces clavuligerus

The following medium produced clavulanic acid when using the conditionsand vegetative seed inoculum as described in Example 1.

Glucose--2%

Lab Lemco (Oxoid)--1%

Oxoid Yeast Extract--0.3%

CaCO₃ --0.3%

Medium prepared in deionised water.

Optimum titres were achieved in 3-5 days and a 1/2500 dilution of theculture filtrate gave 35-45% inhibition in the β-lactamase enzymeinhibition assay.

EXAMPLE 7 Cultivation of Streptomyces clavuligerus

As in Examples 4, 5 and 6 the following medium produced 35-45%inhibition (1/2500 dilution) in the β-lactamase assay at the optimumtitre which is reached 3-5 days after inoculation. All conditions wereas previously described.

Glucose--2% w/v

Arkasoy--1% w/v

CaCO₃ --0.02% w/v

CoCl₂.6H₂ O--0.0001% w/v

Medium prepared in deionised water

EXAMPLE 8 Cultivation of Streptomyces clavuligerus

The following production stage medium when used under standardcultivation conditions as described in previous Examples produced 20-30%inhibition at 1/2500 dilution in the β-lactamase assay between 3-5 daysafter inoculation. Using the paper chromatographic method previouslydescribed, a zone of clavulanic acid was seen at R_(f) 0.46 when culturefiltrate was examined.

Scotasol--2%

Oxoid Yeast Extract--1%

Medium prepared in tap water

Final pH 7.0

EXAMPLE 9 Cultivation of Streptomyces clavuligerus

Under standard cultivation conditions, the following medium producedclavulanic acid 3-5 days after inoculation with the vegetative seed. A1/2500 dilution of the culture gave 20-30% inhibition in the β-lactamaseinhibition assay.

    ______________________________________                                                             g/l                                                      ______________________________________                                        Glycerol               15                                                     Sucrose                20                                                     Proline                2.5                                                    Monosodium Glutamate   1.5                                                    NaCl                   5.0                                                    K.sub.2 HPO.sub.4      2.0                                                    CaCl.sub.2             0.4                                                    MnCl.sub.2 4H.sub.2 O  0.1                                                    FeCl.sub.3 6H.sub.2 O  0.1                                                    ZnCl.sub.2             0.05                                                   MgSO.sub.4 7H.sub.2 O  1.0                                                    Medium prepared in deionised water                                            Final pH 7.1                                                                  ______________________________________                                    

EXAMPLE 10 Cultivation of Streptomyces clavuligerus

A stock Yeatex/glucose agar slope was used to inoculate a Yeatex/glucoseagar slope in a Roux bottle by making a mycelium/spore suspension insterile water. The Roux bottle slope was incubated at 26° C. for 10days. To this slope 100 mls. of sterile water was added and a mycelialsuspension prepared. This was used to inoculate 50 liter of steamsterilised seed medium of the following composition in tap water.

Oxoid Malt Extract--1% w/v

Oxoid Bacteriological Peptone--1% w/v

Glycerol--1% w/v

10% Pluronic L81 Antifoam in Soybean Oil--0.05% w/v

[Pluronic supplied by Jacobs and Van den Berg UK Ltd., 231 The Vale,London, W3 containing a polypropylene-polyethylene block polymer, andSoyabean Oil supplied by British Oil and Cake Mills Ltd., StoneferryRoad, Hull, U.K.].

The medium was contained in a 90 liter stainless steel baffledfermenter, agitated by a 5" vaned disc impeller at 240 r.p.m. Sterileair was supplied at 50 l/min and the tank incubated at 26° C.

After 72 hours, the seed fermenter was used to inoculate 150 liter ofthe same medium using a 5% v/v addition by sterile transfer. Thisproduction stage medium was contained in a 300 L stainless steel, fullybaffled fermenter agitated by a 81/2 vaned disc impeller at 210 r.p.m.Sterile air was supplied at 150 l/min. The fermentation was maintainedat 26° C. Antifoam was added when required in 10 ml. shots (10% PluronicL81 in soyabean oil). Samples were removed for β-lactamase inhibitionassay at regular intervals. The fermenter was harvested between 4-5 daysat the optimum level of β-lactamase inhibitory activity (Table 2).

                  TABLE 2                                                         ______________________________________                                        β-Lactamase Inhibitory Activity of Samples of                            Culture Filtrate taken from a 300 liter                                       Fermentation of Streptomyces Clavuligerus                                     Fermentation                                                                              % Inhibition in β-lactamase                                  Time        Inhibition Assay at a Final                                       (days)      Dilution of 1/2500                                                ______________________________________                                        1.0         12                                                                1.5         20                                                                2.0         31                                                                2.5         36                                                                3.0         50                                                                3.5         54                                                                4.0         51                                                                4.5         56                                                                5.0         55                                                                ______________________________________                                    

EXAMPLE 11 Cultivation of Streptomyces clavuligerus

The seed fermenter was run exactly as described in Example 10 using thesame medium.

After 72 hours, the seed fermenter was used to give a 5% v/v vegetativeinoculum into a 300 liter stainless steel fully baffled fermentercontaining 150 liter of steam sterilised medium agitated by an 81/2 inchvaned disc impeller at 210 r.p.m. Sterile air was supplied at 150 l/min.The fermentation was maintained at 26° C. Antifoam was added whenrequired in 10 ml. shots (10% Pluronic L81 in soya bean oil).

The medium used in the production stage was as described in Example 3with the addition of 0.05% v/v of 10% Pluronic L81/soybean oil antifoamprior to sterilisation.

The β-lactamase inhibitory activity of fermentation samples was similarto those of Example 10 (see Table 2). Paper chromatographic examinationrevealed a zone of clavulanic acid at R_(f) 0.46 using thebioautographic (synergism) method previously described. The size of theclavulanic acid zone increased in parallel with the increase in theβ-lactamase inhibitor assay.

EXAMPLE 12 Cultivation of Streptymces clavuligerus

100 mls of sterile water was added to a sporing culture which had beengrown on Bennetts agar in a Roux bottle for 10 days at 26° C. Amycelium/spore suspension was produced and used to inoculate 75 litersof steam sterilised medium of the following composition in tap water.

Dextrin--2% W/V

Arkasoy 1 50`--1% W/V

10% Pluronic L81 in soyabean oil--0.03% V/V

The pH of the medium was adjusted to 7.0

The medium was contained in a 100 liter stainless steel baffledfermenter, agitated by a 71/2" vaned disc impeller at 140 rpm. Sterileair was supplied at 75 l/minute and the tank incubated for 72 hours at26° C.

The contents of the seed fermenter were used to inoculate 1500 liters ofsteam sterilised medium of the following composition in tap water.

Arkasoy `50`--1.5% W/V

Glycerol--1.0% W/V

KH₂ PO₄ --0.1% W/V

10% Pluronic L81 in soyabean oil--0.2% V/V

The pH of the medium was adjusted to 7.0

The medium was contained in a 2000 liter stainless steel fully baffledfermenter agitated by two 19" vaned disc impellers at 106 r.p.m.

Sterile air was supplied at 1200 liters per minute. Antifoam was addedin 25 ml amounts as required. (10% Pluronic L81 in soyabean oil). Thefermentation was controlled at 26° C. until a maximum yield ofclavulanic acid was obtained between 3-5 days when 200-300 μg/ml ofclavulanic acid were produced.

EXAMPLE 13 Cultivation of Streptomyces clavuligerus

Inoculum was produced in a seed flask as previously described, but usingthe medium described in Example 3 (with pH of the medium adjusted to7.0). This was used to inoculate 500 ml conical flasks containing 100 mlaliquots of the following medium prepared in deionised water andsterilised. The inoculum level was 5%.

Prichem P224--1% W/V

Arkasoy `50`--1.5% W/V

KH₂ PO₄ --0.1% W/V

The pH of the medium was adjusted to 7.0

The inoculated flasks were shaken at 26° C. and optimum β-lactamaseinhibitory activity was achieved between 3-5 days. Levels of 300-500μg/ml of clavulanic acid were achieved.

Prichem P224 is a triglyceride supplied by Prices Limited, Bromborough,Bebington, Wirral, Cheshire, U.K.

Prichem P.224 is based on oleic acid (65%), palmitic acid (11%) andother similar acids.

EXAMPLE 14 Isolation of Crude Clavulanic Acid Sodium Salt

Harvested culture liquor produced as described in Example 10 wasclarified by continuous flow centrifugation and the mycelium discarded.From 150 liter of fermentation liquor 120 liter of clarified culturefluid was obtained. This filtrate gave 58% inhibition in the β-lactamaseinhibition assay at 1/2500. The filtrate was chilled to 5° C. and 40liter of n-butanol added. The mixture was stirred and 25% H₂ SO₄ addeduntil the pH was 2.0. The acidified mixture was stirred for a further 10mins. before separating the phases by centrifugation. The aqueous phasewas discarded. To the n-butanol extract 0.5% of Norit GSX carbon wasadded and the mixture stirred for 15 minutes. The carbon was discardedafter removal by filtration using a diatomaceus earth as a filter aid.To the n-butanol a 1/4 volume of deionised water was added and themixture stirred while adding 20% NaOH solution until the pH hadequilibated at 7.0. The phases were separated by centrifugation and then-butanol phase discarded. The aqueous phase was concentrated underreduced vacuum to 800 ml. and then freeze dried. This yielded 35 g. of acrude solid preparation of clavulanic acid with an I₅₀ of 1.3 μg/ml inthe β-lactamase inhibition assay. This solid preparation was stored dryat -20° C. while awaiting further purification.

EXAMPLE 15 Isolation of Crude Clavulanic Acid Sodium Salt

One liter of culture filtrate giving 53% inhibition at 1/2500 in theβ-lactamase inhibition assay and obtained as described in Example 12 waspercolated down a 1 inch diameter×6 inch column of Permutit Isoporeresin FF 1P (SRA 62) in the Cl⁻ form [supplied by Permutit Co. Ltd.,632-652 London Road, Isleworth, Middlesex, U.K.]. The culture filtratewas followed by 300 ml. of distilled water to wash the column. Elutionof the active β-lactamase inhibitor was achieved with 0.2M NaClsolution. Fractions (20 ml.) were collected and assayed at a 1/2500final dilution in the β-lactamase inhibition assay. Active fractionswere combined and concentrated under vacuum to 20 ml. This solution wasdesalted by gel exclusion chromatography on a Biorad Biogel P2 column11/2 inches in diameter with a gel bed of 16 inches and eluted with 1%n-butanol in water. [Biogel P2 is supplied by Bio Rad Laboratories, 32nd and Griffin Ave., Richmond, Calif., U.S.A.]. The active fractions, asdetermined by the β-lactamase inhibition assay, were combined. Sodiumchloride eluted after clavulanic acid and was detected using silvernitrate solution. The combined active fractions were concentrated andfreeze dried.

One liter of culture filtrate after the above treatment yielded 0.45 g.of a crude solid preparation of clavulanic acid having an I₅₀ of 0.92μg/ml.

This solid was stored at -20° C. while awaiting further purification.

EXAMPLE 16 Isolation of Crude Clavulanic Sodium Salt

Culture filtrate containing 300 μg/ml of clavulanic acid is acidifiedusing an in-line mixer system, extracted with n-butanol and clavulanicacid is back extracted into water at neutral pH.

Chilled culture filtrate (5°-10° C.) was pumped to an in-line mixer atthe inlet of which, enough 6% (v/v) nitric acid was added to maintain anoutlet pH of 2.0±0.1. The acidified filtrate was passed at 4.1/minthrough a glycol cooled plate heat exchanger (A.P.V. Ltd.) to maintain atemperature between 2°-5°. The pH was monitored in a flow cell beforepassing into a three stage counter current separator (WestfaliaSeparator Ltd., Model EG 1006).

Chilled water saturated n-butanol (at about 5° C.) was pumped at 3 l/mininto the counter current separator.

The aqueous outlet from the counter current separator was run to waste.Entrained water was removed from the butanol outflow of the countercurrent separator using a liquid/liquid centrifugal separator. (AlfaLaval Ltd. Model 3024X-G). The butanol was collected in a stainlesssteel vessel fitted with a cooling jacket in which it was stored atabout 5° C.

From the vessel, 40 l aliquots were removed and thoroughly mixed with 2l of chilled water (5° C.), saturated with n-butanol. The pH of thismixture was adjusted to pH 6.8±0.1 using 20% sodium hydroxide solution.

This aqueous extract/butanol mixture was fed to a liquid/liquidcentrifugal separator (Sharpless Centrifuge Ltd. Model M35PY-5 PH) at apumped rate of 2 l/min.

From 1800 l of culture filtrate, 90 l of aqueous phase was recovered,containing 39% of the clavulanic acid present in the culture filtrate.

15 l of the aqueous extract was adjusted from 2%, to 8%, total solids bythe addition of 60 g sodium chloride per liter, and spray dried(Anhydro, Copenhagen, Type Lab S 1). The conditions used were: Feed rate2 l/hr Atomizer voltage 170 v; Heater setting 6-7; Inlet temp 150° C.;Outlet temp 80° C.

The dried product, total weight 1 kg., contained 62% of the clavulanicacid present in the feedstock.

The remaining 75 l of aqueous extract was concentrated byultrafiltration (De Danske Sukkerfabrikker. Laboratory Module, MembraneType 900). The operating procedure was to re-circulate the retentatefrom a stainless steel tank, fitted with a cooling system, with theoutlet valve set so as to give a differential pressure across the 40membranes of 25 atmospheres. The temperature was maintained at 2°-5° C.and the pH at 6.8±0.1 by addition of 2N hydrochloric acid, as necessary.The volume was reduced to 34 l which contained 72% of the clavulanicacid present in the feedstock.

The aqueous concentrate was stored at about 5° C., adjusted to 8%solids, and spray dried as above. The dried material contained 75% ofthe clavulanic acid present in the feedstock to the spray drier.

The total spray dried product, from the 90 l of aqueous extractcontained 69.4 g of clavulanic acid which was 72% of the clavulanic acidin the spray drying feedstock and 21% of the clavulanic acid present inthe 1800 l of culture filtrate.

EXAMPLE 17 Partial Purification of Crude Clavulanic Acid

Crude clavulanic acid preparations obtained as described in Example 15were purified by ion exchange chromatography. Eighteen grams of materialprepared as described in Example 15 having an I₅₀ value of 1.3 μg/ml(final concentration) were dissolved in 25 ml. of distilled water andapplied to a 11/2"×16" bed of Permutit FF 1P (SRA 62) resin in thechloride form. The column was eluted with a sodium chloride gradientformed by gravity feeding 0.5M sodium chloride into a mixing reservoircontaining 1 liter of distilled water which in turn fed thechromatographic column. 10 ml. cuts were collected and β-lactamaseinhibitory activity assayed using a 1/2500 dilution of the fractions.Activity was eluted after a main band of colour between fractions 24 and30. The active fractions were combined and concentrated to 30 ml.

This solution was desalted using 2"×18" bed of Biorad Biogel P2 andeluting with 1% n-butanol in water. The 20 ml. fractions were assayedfor clavulanic acid content using the β-lactamase inhibition assay. Thefractions were also spotted onto paper strips and sprayed with eitherthe Ehrlich or the triphenyltetrazolium spray reagents described inDescription 3. β-lactamase inhibitory activity correlated with the pinkor red spots respectively produced by these reagents. Active cuts werecombined, excluding those containing sodium chloride and concentratedunder vacuum to dryness. This yielded 520 mg. of partially purifiedclavulanic acid sodium salt with an I₅₀ of 0.2 μg/ml in the standardβ-lactamase inhibitor assay.

Thin layer chromatography (silica gel) of this clavulanic acidpreparation gave the following R_(f) values: n-butanol/ethanol/water4:1:5 v/v top phase R_(f) 0.37; n-butanol/acetic acid/water 12:3:5 v/vR_(f) 0.44; isopropanol/water 7:3 v/v R_(f) 0.78. The zones weredetected by spraying with Ehrlich's reagent. 6-Aminopenicillanic run asa marker and detected with the same spray had R_(f) values of 0.38; 0.39and 0.77 respectively.

EXAMPLE 18 Partial Purification of Clavulanic Acid Sodium Salt

Culture filtrate produced as described in Example 12 was solventextracted as in Example 14 to give a solid preparation which was furtherpurified by ion exchange chromatography using Whatman diethylaminoethylcellulose DE 52. This solid (10 g.) was dissolved in 20 ml. of distilledwater and applied to a 11/2"×20" column of DE 52 cellulose previouslyequilibrated with 0.01M sodium phosphate buffer pH 7.5. The column waseluted with a NaCl gradient. 0.1M NaCl in 0.01M sodium phosphate bufferpH 7.5 was fed into a mixing chamber containing 1 liter of 0.01Mphosphate buffer pH 7.5 which in turn was connected to the column.Fractions (10 ml.) were collected and these were assayed for β-lactamaseinhibitory activity at a dilution of 1/2500. The fractions were alsoexamined for antibacterial activity by the hole-in-plate assay methodusing nutrient agar plates seeded with Klebsiella aerogenes. Thefractions having the highest β-lactamase inhibitory activity and givingzones of inhibition in the hole-in-plate assay were combined,concentrated and then desalted on a Biorad Biogel P2 column. Thesefractions were shown to contain clavulanic acid by paper and thin layerchromatography.

EXAMPLE 19 Isolation of Solid Clavulanic Acid Sodium Salt

A partially purified solid preparation of clavulanic acid (500 mg)prepared as in Example 17 was loaded onto a Whatman microcrystallineCC.31 cellulose column with 1"×20" bed size. The chromatographic solventwas n-butanol/ethanol/water 4:1:5 v/v, top phase. The column was run at4° C. and 4 ml. fractions collected. Fractions were tested for thepresence of clavulanic acid by spotting onto filter paper and sprayingwith the Ehrlich (pink spot) or triphenyltetrazolium (red spot) sprayreagents. These spot tests were confirmed by β-lactamase inhibitionassays at a 1/1250 dilution. Active fractions were combined and driedunder vacuum on a rotary evaporator. The solid was dissolved in a smallvolume of distilled water and freeze dried. A white solid preparation ofthe sodium salt of clavulanic acid was obtained (40 mg) having an I₅₀ of0.08 μg/ml in the β-lactamase inhibition assay.

EXAMPLE 20 Isolation of Solid Clavulanic Acid Sodium Salt

Concentrated back extract (6 l) (from ultrafiltration in Example 16)containing 10 g of clavulanic acid as determined by the β-lactamaseinhibition assay of Description 1. This was percolated at 1 l/hr onto a2"×24" column of Permutit Zerolit FF 1 P SRA 62 anion exchange resin inthe chloride form. The column was then washed with 2 l of deionizedwater prior to elution with a sodium chloride gradient. The gradient wasformed by a reservour containing 4 l of 1.4 m NaCl feeding astirred-reservoir containing 4 l of 0.7 NaCl which in turn was connectedto a stirred reservoir containing 4 l of deionized water which wasconnected via a pump to the column. The column was eluted at 2.5 ml/minand 25 ml fractions collected. Fractions were assayed by the β-lactamaseinhibition assay.

Active fractions (nos. 140-230) were combined and vacuum evaporated tonear dryness. Ethanol (500 mls) was then added and the solid filteredoff after vigorous shaking. The ethanol extract was then vacuumevaporated to dryness on a rotary avaporator and redissolved indeionized water (40 mls). This was loaded onto a 4"×24" column of BioradBiogel P₂ and eluted with a 1% n-butanol solution. Fractions werecollected (25 ml) and assayed for β-lactamase inhibitory activity at a1/2500 final dilution. Tests for sodium chloride content on 1/25dilutions of the fractions were made using silver nitrate solution.Those fractions containing clavulanic acid free of sodium chloride werecombined, concentrated by evaporation of the solvent under reducedpressure to 20 mls and then freeze dried. This yielded 4.8 g of thesodium salt of clavulanic acid. (I₅₀ about 0.06 μg/ml).

EXAMPLE 21 Preparation of an Ester of Clavulanic Acid (Methyl Ester)##STR12##

19.8 mg. of the sodium salt of clavulanic acid was dissolved in 0.5 ml.dry dimethylformamide and treated with 0.25 ml. methyl iodide. Afterstanding at room temperature for 1.5 hours under anhydrous conditions,the solvents were removed in vacuo. The residue was purified by P.L.C.on silica gel (Kieselgel 60F254 supplied by E. Merck, Darmstadt,Germany) eluting with ethyl acetate to give clavulanic acid methyl esteras a colourless oil (R_(f) 0.38; red colour with triphenyltetrazoliumchloride spray) which had the following properties:

Analysis: Found C 50.49 H 5.43 N 6.29. C₉ H₁₁ NO₅ Requires C 50.70 H5.20 N 6.57.

λmax (Methanol): no absorption >215 nm.

νmax (Film): 3300-3600 (Broad), 1800, 1750, 1695 cm⁻¹.

Approximate 1st order N.M.R. (CDCl₃): 2.49 (broad S, 1, exchanged withD₂ O), 3.05 (d, 1, J=17.5 Hz), 3.54 (dd, 1, J=17.5 Hz, J₂ =2.5 Hz), 3.84(S, 3). 4.24 (d, 2, J=7 Hz), 4.93 (dt, 1, J=7 Hz, J₂ =1.5 Hz), 5.07 (d,1, J=1.5 Hz), 5.72 (d, 1, J=2.5 Hz).

Molecular weight (mass spectrum): 213.0635.

Calculated for C₉ H₁₁ NO₅ : 213.0637.

Thin layer chromatography of the methyl ester showed a single zone ineach of the following solvent systems; butanol/ethanol/water 4:1:5 v/vtop phase R_(f) 0.75; isopropanol/water, 7:3 v/v R_(f) 0.95;ethylacetate/ethylalcohol 8:2 v/v R_(f) 0.87. The zones were detected bybioautography using Klebsiella aerogenes with added benzylpenicillin(synergism system).

EXAMPLE 22 Preparation of an Ester of Clavulanic Acid (p-nitrobenzylester) ##STR13##

Treatment of the sodium salt of clavulanic acid with p-nitrobenzylbromide in dry DMF gave, after P.L.C., a colourless oil whichcrystallised from chloroformether to give to p-nitrobenzyl ester ofclavulanic acid as white feathery needles, m.p. 111°-112° C., which onrecrystallisation had a mp of 117.5°-118° C.

EXAMPLE 23 Preparation of an Ester of Clavulanic Acid (Benzyl Ester)##STR14##

Impure3-(β-hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylicacid sodium salt (thought to be roughly 55 mg. of pure material) in drydimethylformamide (0.64 ml.) was treated with benzyl bromide (0.18 ml.).The solution was kept at room temperature (approx. 17°-18° C.) for 3hours under anhydrous conditions. The reaction mixture was fractionatedon silica gel, eluting with ethyl acetate, to give in substantially pureform the benzyl ester or3-(β-hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylicacid 63 mg.) as a colourless oil. i.r. (film) 1800, 1745, 1695 cm⁻¹ ;n.m.r. (CDCl₃), 2.25 (s,1, exchangeable with D₂ O), 3.05 (d,1,J=17 Hz),3.51 (dd,1,J=17 Hz, J₂ =2.5 Hz), 4.24 (d,2,J=7.5 Hz), 4.92 (dt,1,J=7.5Hz, J₂ =1.5 Hz), 5.15 (d,1,J=1.5 Hz), 5.24 (s,2), 5.71 (d,1,J=2.5 Hz),7.45 δ (s,5).

EXAMPLE 24 Preparation of the Benzyl Ester of Clavulanic Acid from CrudeExtracts of the Fulture Filtrate of S. Clavuligerus

Culture filtrate 20 l. obtained as described in Example 10 was vacuumevaporated using a climbing film evaporator to 5 l. The concentrate wasthen freeze dried using an Edwards E.F.6 shelf freeze-drier manufacturedby Edwards High Vacuum Ltd. The 300 g. of solid so obtained contained 3g. of sodium Clavulanic acid as determined by the enzyme inhibitionassay. The solid was suspended in 900 ml. of dry dimethylformamide and150 ml. of benzyl bromide was added. The mixture was stirred for 2 hoursat room temperature and then diluted with 1 l. of ethyl acetate. Thereaction mixture was filtered and the filtrate concentrated to as low avolume as was possible. The oily residue was extracted with a further 1l. of ethyl acetate and the extract filtered. The filtrate was againconcentrated and the resulting oily residue loaded onto a 3"×14" silicagel column (Biogel Biosil A 100 mesh) in cyclohexane. The column waseluted with cyclohexane to remove benzyl bromide and the solvent wasthen changed to ethyl acetate and 20 ml. fractions collected. Fractionswere tested for the presence of the benzyl ester of clavulanic acid byspotting onto glass backed silica gel t.l.c. plates (Merck precoatedsilica gel 60 F 254) and spraying with 2,3,5-triphenyl-tetrazoliumchloride (TTC) spray reagent. Fractions giving intense red spots withthis reagent were further examined by t.l.c. on silica gel plates usingchloroform-ethyl acetate 8:2 as the solvent and spraying the developedplates with TTC spray. The benzyl ester of clavulanic acid runs at R_(f)0.31 at 22° C. Fractions containing this ester were combined andconcentrated to 15 ml. and this solution was further chromatographed ona 11/2"×16" silica gel column (Merck silica gel H, type 60) withchloroform/ethyl acetate 8:2 as the solvent. 15 ml. fractions werecollected and tested for the benzyl ester as described above. Thosefractions containing the ester were concentrated to 8 ml. and finallypurified by column chromatography on a 1"×16" silica gel column (Mercksilica gel H, type 60) with ethyl acetate cyclohexane 8:2 as thesolvent. Selected fractions were combined and vacuum evaporated to givepure benzyl ester as an oil, 160 mg.

EXAMPLE 25 Preparation of clavulanic acid benzyl ester

Spray dried solid (3.3 kg) containing 69.4 g of clavulanic acid asdetermined by enzyme inhibition assay was obtained as described inExample 16. The solid was slurried in 5.5 l. of dimethylformamide and500 mls. of benzyl bromide added. After stirring at room temperature for2 hours, 12 l. of ethyl acetate were added and the solids removed byfiltration. The filtrate was vacuum evaporated to an oily residue (212g). The residue was loaded onto a column containing a 4"×13" bed ofsilica gel (Hopkins & Williams NPC) in cyclohexane. The column waseluted with 12 l. of cyclohexane to remove excess benzyl bromide. Theeluent was then changed to ethyl acetate and 500 ml. fractionscollected. These were tested for benzyl clavulanate content by spottingonto silica gel t l c plates (Merck precoated silica gel 60 F 254) andspraying with 2,3,5 triphenyltetrazolium chloride (TTC) spray reagent.Fractions giving intense red spots were further examined by t l c onsilica gel with chloroform/ethyl acetate 8:2 as the solvent and sprayingthe developed plates with T T C spray. Fractions 5-13 contained the bulkof the ester, and these were combined and vacuum concentrated to an oil(79.3 g). This preparation was then chromatographed on a 4"×18" columnof silica gel (Merck silica gel H type 60) with chloroform/ethyl acetate8:2 as the solvent. Fractions were selected as described above andyielded on concentration 45.9 g of oil which was of 62% purity asadjudged by NMR spectroscopy.

This product was finally chromatographed on a 23/4"×18" column ofSephadex LH 20 in cyclohexane/chloroform 1:1. After selection offractions and concentration a colourless oil (27.6 g) was obtained whichproved to be 95% pure benzyl ester of clavulanic acid as determined byNMR spectroscopic examination.

(Sephadex LH20 is a hydroxypropyl derivative of Sephadex Q25 supplied byPharmacia Great Britain, 75 Uxbridge Road, London, W.5, U.K.).

EXAMPLE 26 Preparation of clavulanic acid benzyl ester

Culture filtrate (150 l) pH 7.0 contained 16.2 g. of clavulanic acid(sodium salt) as determined by the enzyme inhibition assay. Thisfiltrate was stirred with 5 kg. of Amberlyst A.26 anion exchange resinin the chloride form (Rohm & Haas Company, Philadelphia, USA) for 1 hourat room temperature. The resin was then filtered and the filtratereassayed, showing that 6.4 g of clavulanic acid had been removed. Theresin was washed with 20 l. of deionised water followed by 20 l. ofacetone and 10 l. of dimethyl formamide (DMF). After refiltering theresin was suspended in 2.3 l. of DMF/0.2M NaI. To this was added 200mls. of benzyl bromide and the suspension stirred thoroughly. Afterstanding at room temperature for 16 hours, ethyl acetate (2 l) wasadded, and the resin then filtered, further washings (Ethyl acetate) ofthe resin were combined with the filtrate. The extract was thenconcentrated to a small volume and chromatographed on 3"×18" silica gelcolumn (Merck silica gel H type 60) with ethyl acetate/cyclohexane 8:2as the solvent. Fractions containing benzyl clavulanate were selected byspotting onto silica gel t l c plates and spraying with TTC reagent asdescribed previously (Example 24). Those selected were concentrated to20 mls and then chromatographed on a 11/2"×18" silica gel column (Mercksilica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent.Selected fractions were combined and evaporated to a colourless oil (440mgs) which was 90% benzyl clavulanate as determined by NMR spectroscopy.

EXAMPLE 27 Preparation of the benzyl ester of clavulanic acid from crudeextracts of the culture filtrate of S. Clavuligerus

An aliquot of aqueous back extract of the butanol extract of culturefiltrate obtained as described in Example 14 was freeze dried using anEdwards chamber drier. A 24 g. portion of the solid obtained contained0.96 g. of sodium clavulanic acid as determined by the enzyme inhibitionassay. This solid was suspended in 75 ml. of dry dimethylformamide and75 ml. of benzyl bromide was added. The mixture was stirred for 2 hoursat room temperature. The suspension was then diluted with 500 ml. ofethyl acetate and the mixture filtered. The filtrate was concentrated toan oily residue on a vacuum rotary evaporator. This residue was loadedonto a 2"×14" silica gel column (Biogel Biosil A.100 mesh) incyclohexane. Benzyl bromide was eluted from the column and then thesolvent was changed to ethyl acetate and 10 ml. fractions was collected.Fractions containing the benzyl ester of clavulanic acid were selectedas in Example 24. Further purification was also achieved as described inExample 24 by column chromatography. This process yielded 220 mg. ofpure benzyl ester.

EXAMPLE 28 Preparation of clavulanic acid sodium salt ##STR15##

Substantially pure benzyl clavulanate (281 mg) in ethanol (25 ml.)containing sodium hydrogen carbonate (82 mg.) was hydrogenated over 10%Pd/C (90 mg.) for 25 minutes at room temperature and atmosphericpressure. The catalyst was filtered off, washed with water and ethanol,and the combined filtrates evaporated under reduced pressure at roomtemperature. The residual semi-solid was triturated with acetone,filtered and washed with ether to yield sodium clavulanate (135 mg.).

EXAMPLE 29 Hydrolysis of clavulanic acid methyl ester to clavulanic acid

2.17 mg. of clavulanic acid ester was dissolved in 0.1 ml. methanol andtreated with 0.208 ml. sodium hydroxide solution (0.0482N). After 1 hourat room temperature, the reaction mixture contained several products.T.L.C. analysis indicated that one of the major components had an R_(f)identical to that of the sodium salt of clavulanic acid; colourreactions and biological assay were consistent with this component beingthe sodium salt of clavulanic acid.

Slow conversion of the ester to clavulanic acid was seen when 1 mg/ml.of the compound was incubated at 37° C. in 0.05M phosphate buffer at pH7. The reaction was followed by paper chromatography (bioautographicsystem). Using the butanol/ethanol/water system to follow the reactionover a period of 2 hours the zone of the methyl ester at R_(f) 0.79decreased in size as the zone of clavulanic acid at R_(f) 0.12increased.

EXAMPLE 30 Antibacterial spectrum of clavulanic acid

The antibacterial activity of clavulanic acid sodium salt against arange of bacteria was determined using the microtitre method. Serialdilutions of clavulanic acid sodium salt in Oxoid sensitivity test brothcontained in a microtitre plastic tray were inoculated with an overnightbroth culture of each organism so that the final dilution of theinoculum was 0.5×10⁻⁴. The cultures were incubated overnight and thepoints of bacterial growth recorded next morning by observing theturbidity of the culture. The results, expressed as approximate MICvalues (minimum inhibitory concentration μg/ml.) are recorded in Table 3which shows that the compound has a broad spectrum of antibacterialactivity.

                  TABLE 3                                                         ______________________________________                                        ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID                                     SODIUM SALT                                                                                       Minimum Inhibitory                                        Bacterial Strain    Concentration μg/ml.                                   ______________________________________                                        Staphylococcus aureus (Oxford H)                                                                  7.5                                                       Staphylococcus aureus (Russell)                                                                   7.5                                                       Bacillus subtilis   62                                                        Streptococcus faecalis                                                                            >500                                                      Streptococcus pyogenes CN 10                                                                      125                                                       Escherichia coli NCTC 10418                                                                       31                                                        Klebsiella aerogenes                                                                              31-62                                                     Klebsiella oxytocum 62                                                        Enterobacter aerogenes T 624                                                                      31                                                        Enterobacter cloacae                                                                              62                                                        Acinetobacter anitratus                                                                           125                                                       Providentia stuartii                                                                              125                                                       Serratia marcescens 125                                                       Proteus mirabilis C977                                                                            62                                                        Proteus vulgaris W090                                                                             31                                                        Salmonella typhimurium                                                                            31                                                        Shigella sonnei     62                                                        Pseudomonas aeraginosa A                                                                          500                                                       ______________________________________                                    

EXAMPLE 31 Examples of β-lactamase inhibition by clavulanic acid sodiumsalt

Clavulanic acid progressively and irreversibly inhibits the β-lactamaseof Escherichia coli. The method of Description 1 shows that the otherβ-lactamases shown in Table 4 are also inhibited by clavulanic acid.

                  TABLE 4                                                         ______________________________________                                        INHIBITION OF β-LACTAMASES BY CLAVULANIC ACID                                              Approximate                                                                   I.sub.50 Value Relative to                                  Source of β-lactamase                                                                      Escherichia coli JT 4 = 1                                   ______________________________________                                        Staphylococcus aureus (Russell)                                                                 1.0                                                         Escherichic coli JT4                                                                            1.0                                                         Escherichia coli B11                                                                            2.0                                                         Klebsiella aerogenes A                                                                          0.6                                                         Pseudomonas aeruginosa 1822                                                                     5.0                                                         (R factor)                                                                    Pseudomonas dalgleish                                                                           0.1                                                         ______________________________________                                    

With penicillin G as substrate the I₅₀ of clavulanic acid sodium saltagainst the β-lactamase of Staph. aureus (Russell) is approximately 0.06μg/ml.

EXAMPLE 32 Examples of activity of clavulanic acid methyl ester

Tests for antibacterial activity in broth showed clavulanic acid methylester to have broad spectrum activity but of a lower order than shown byclavulanic acid. It was not clear whether this activity was the activityof the compound itself or of clavulanic acid liberated by slow aqueoushydrolysis of the ester. Clavulanic acid methyl ester showed markedantibacterial synergism in combination with ampicillin or cephaloridineagainst bacteria resistant to these antibiotics. Thus, the minimuminhibitory concentration (M.I.C.) for ampicillin against Staphylococcusaureus (Russell) was reduced from 500 μg/ml. to <0.4 in the presence of1.0 μg/ml. clavulanic acid methyl ester. The M.I.C. for cephaloridinewas reduced from 1.5 μg/ml. to <0.03 μg/ml. in the presence of 1 μg=/ml.of clavulanic acid methyl ester. The M.I.C. for ampicillin againstProteus mirabilis C889 was reduced from 500 μg/ml. to 31 μg/ml. in thepresence of 5 μg/ml. clavulanic acid methyl ester.

EXAMPLE 33 Preparation pivaloyloxymethyl clavulanate

To a stirred solution of bromomethyl pivalate (C.37 g) in drydimethylformamide (5 ml) was added sodium clavulanate (0.49 g). After 2hrs. at room temperature the reaction mixture was treated with ethylacetate (20 ml), cyclohexane (10 ml) and water (20 ml). The mixtureseparated into two layers and the non-aqueous layer was separated,washed with water (20 ml) and dried over sodium sulphate. The driedsolution was evaporated to leave the required product as a pale yellowoil. (500 mg). N.m.r. (CDCl₃), 1.26 (s,9), 3.13 (d,1,J=17 Hz), 3.62(dd,1,J,=17 Hz, J₁ =2.5 Hz, 4.3(d,2,J=7.5 Hz), 5.0 (dt,1,J=7.5 Hz, J₂=1.5 Hz), 5.16(d,1,J=1.5 Hz), 5.79(d,1,J=2.5 Hz), 5.92δ(s,2);i.r.(liquid film), ν β-lactam C.O 1800 cm⁻¹, ester C═O 1760 cm⁻¹.

EXAMPLE 34 Preparation of clavulanic acid phthalide esters

To a stirred solution of 3-bromophthalide (0.43 g) in drydimethylformamide (5 ml) was added sodium clavulanate (0.5 g) and thesolution was left at room temperature for 2 hours. The solution wastreated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (30ml) and shaken thoroughly. The non-aqueous layer was washed with water(20 ml), dried (Na₂ SO₄) and evaporated to yield a pale yellow gum. Thetwo diastereomeric esters were separated using high pressure liquidchromatography on a 40 cm×10 mm column of silica gel (Merckosorb SI 60,5μ) eluting with ethyl acetate at a flow rate of 3 ml/min.

The first phthalide ester (retention time 7.15 min) crystallised fromethyl acetate as needles, mp 102°, and had the following i.r. (nujolmull) ν β-lactam C═O 1790 cm⁻¹ ester C═O 1755 cm⁻¹ n.m.r. (CD₃ COCD₃):3.14 (d,1,J=17.5 Hz), 3.76 (dd,1,J,=17.5 Hz, J₂ =2.5 Hz), 4.25(d,2,J=7.5Hz), 5.0 (dt,1,J₁ =7.5 Hz, J₂ =1.5 Hz), 5.4 (s,1,J=1.5 Hz),5.82(d,1,J=2.5 Hz), 7.7 (s,1), 8.06δ(m,4); M.wt (mass spectrometry: 331.0696corresponds to C₁₆ H₁₃ NO₇ (calc. 331.0692). The second diasterioisomer(retention time 8.85 min) had the following i.r. (CH₂ Cl₂ solution) νβ-lactam C═O 1800 cm⁻¹, ester C═O 1780 cm⁻ 1 ; nmr (CDCl₃) 2.42 (broadS,1, exchangeable with D₂ O), 3.12 (d,1,J=18 Hz), 3.60 (dd,1,J₁ =18 Hz,J₂ =2.5 Hz), 4.30 (d,2,J=7.5 Hz), 5.0 (dt,1,J₁ =7.5 Hz, J₂ =1.5 Hz),5.12 (d,1,J=1.5 Hz), 5.76 (d,1,J=2.5 Hz), 7.52 (S,1), 7.85δ(m,4).##STR16##

EXAMPLE 35 Preparation of nonyl clavulanate

Sodium clavulanate (44 mg) in dry dimethylformamide (2 ml) was treatedwith nonyl iodide (76 mg) and left at room temperature for 2 hours. Thesolution was evaporated and the residue fractionated on silica gel,eluting with ethyl acetate-hexane (2:1) to give the product as an oil;i.r.(film) 1800, 1745, 1690 cm⁻¹. M.wt. (mass spectrometry)=325.1890which corresponds to C₁₇ H₂₇ NO₅. (calc. 325.1889).

EXAMPLE 36 Preparation of clavulanic acid

Benzyl clavulanate (100 mgs) in ethanol (5 ml) was hydrogenated over 10%Pd/C (30 mgs) for 45 minutes at ambient temperature and atmosphericpressure. The catalyst was filtered, washed with ethanol and thecombined filtrates were evaporated in vacuo to give clavulanic acid asan unstable, viscous oil (58 mgs). N.m.r. (C₅ D₅): 3.05(d,1,J=18 Hz),3.60(dd,1,J₁ =18 Hz), J₂ =2.5 Hz), 4.75(d,2,J=7.5 Hz), 5.58(t,1,J=7.5Hz),5.66 (S,1), 6.0δ(d,1,J=2.5 Hz).

EXAMPLE 37 Preparation of methyl clavulanate

Clavulanic acid (130 mgs) in ethanol (10 ml) was treated with excessdiazomethane in ether. After 2 minutes at room temperature the reactionwas shown (t l c) to be complete. The solution was evaporated in vacuoand the residue purified by chromatography on silica gel, eluting withethyl acetate. The fractions containing methyl clavulanate were combinedand evaporated to give a clear oil (104 mgs).

EXAMPLE 38 Preparation of methyl clavulanate

Clavulanic acid (200 mgs) in acetonitrile (5 ml ) was cooled and stirredat 0°. Methanol (0.5 ml) and then dicyclohexyldicarbodiimide (206 mg.)were added and the reaction mixture was stirred at room temperatureovernight. The suspension was filtered and the filtrate evaporated invacuo to give crude methyl clavulanate. The crude product was purifiedby chromatography on silica gel, eluting with ethyl acetate, to give aclear oil (140 mg).

EXAMPLE 39 Preparation of phenyl clavulanate

Clavulanic acid (100 mg) in acetonitrile (5 ml) was cooled and stirredat 0°. To the solution was added phenol (0.94 g) anddicyclohexyldicarbodiimide (100 mg) and the reaction mixture was stirredat room temperature overnight. The suspension was filtered and thefiltrate evaporated. The residue was fractionated on silica gel, elutingwith ethyl acetate-hexane (1:1) to give phenyl clavulanate (70 mg). I.r.(film) 1800, 1770, 1690 cm⁻¹. N.m.r. (CDCl₃) 2.18 (broad s,1), 3.06 (dd,1,J=17 Hz,J₂ =0.9 Hz), 3.54 (dd,1,J₁ =17 Hz,J₂ =2.6 Hz), 4.29 (d,2,J=7.5Hz), 5.1(dt,1,J₁ =7.5 Hz,J₂ =1.5 Hz) 5.29 (d,1,J=1.5 Hz),5.76(dd,1,J₁=2.6 Hz,J₂ =0.9 Hz), 7.35 δ(m,5). M.wt. (mass spectrometry)=275.0777which corresponds to C₁₄ H.sub. 13 NO₅ (calc. 275.0794).

EXAMPLE 40 Preparation of 2,2,2-trichloroethyl clavulanate

Sodium clavulanate (221 mgs) was suspended in dry tetrohydrofuran (5mls) and stirred at 0°. Trichloroethylchloroformate (211 mg) in drytetrohydrofuran (1 ml) was added to the above suspension over 20minutes. The mixture was allowed to reach room temperature and stirredovernight. The suspension was filtered and the filtrate evaporated invacuo. The residue was chromatographed on silica gel eluting with ethylacetate--hexane (2:1) to give the resulting product as an oil, i.r(film) 1800,1760,1690 cm⁻¹. n.m.r. (CDCl₃) 1.56 (broad S,1), 3.07(dd,1,J₁ =17.5 Hz,J₂ =0.7 Hz), 3.56 (dd,1,J₁ =17.5 Hz, J₂ =2.5 Hz), 4.24(d,2,J=7.5 Hz), 4.69 (d,1,J=12 Hz), 4.92 (d,1,J=12 Hz), 5.02 (dt,1,J₁ =5hz, J₂ =1.3 Hz), 5.19(d,1,J=1.3 Hz), 5.73δ(dd,1,J₁ =2.5 Hz, J₂ =0.7 Hz).M.wt. (mass spectrometry)=328.9621 which corresponds to C₁₀ H₁₀ NO₅ Cl₃(calculated 328.9625). ##STR17##

EXAMPLE 41 Preparation of sodium clavulanate

Benzyl clavulanate (840 mgs) in ethanol (30 ml) and water (5 ml) washydrogenated over 10% Pd/C (267 mgs) and sodium bicarbonate (244 mgs)for 25 minutes at room temperature and atmospheric pressure. Thecatalyst was filtered, washed with water and ethanol and the combinedfiltrates were evaporated in vacuo. The product crystallised from awater-acetone mixture as microneedles (565 mgs). Recrystallisation fromwater-acetone gave needles which, after drying over P₂ O₅ in vacuo for24 hours gave the following analysis: C 41.01, 40.86; H 3.77, 3.64; N5.68, 5.51; i.r.(KBr disc) 1785, 1700, 1620 cm⁻¹ ; Nmr (D₂ O) 3.06(d,1,J=18.5 Hz), 3.57 (dd,1,J₁ =18.5 Hz, J₂ =2.5 Hz), 4.15 (d,2,J=8 Hz),5.3 (HOD), 4.9(m), 5.71 (d,1,J=2.5 Hz).

EXAMPLE 42 Antibacterial synergism between ampicillin and clavulanicacid sodium salt

The minimum inhibitory concentration (M.I.C. values) of ampicillin,clavulanic acid sodium salt and ampicillin in the presence of 1 μg/ml.clavulanic acid sodium salt were determined for a range of β-lactamaseproducing bacteria. The organisms were inoculated into Oxoid sensitivitytest broth located in small wells in a plastic tray and containingseparate concentration gradients of ampicillin, clavulanic acid sodiumsalt or ampicillin plus 1 μg/ml. clavulanic acid sodium salt (microtitremethod). The final dilution of the overnight broth inoculum was0.5×10⁻². The tray was incubated at 37° C. overnight and a record madenext morning of the end points of bacterial growth. The M.I.C. values inμg/ml. are recorded in Table 5 which reveals that the synergist at thelow concentration of 1 μg/ml. markedly enhances the antibacterialactivity of ampicillin against certain gram+ve and gram-ve bacteria. Themechanism of this synergism is likely to involve inhibition ofampicillin destroying β-lactamase enzymes but the existence of othermechanisms cannot be excluded.

Similar results to those shown in Table 5 were obtained when ampicillinwas replaced by amoxycillin or by the phthalidyl ester of ampicillin.

                  TABLE 5                                                         ______________________________________                                        ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN                                    AND CLAVULANIC ACID SODIUM SALT                                                         Minimum Inhibitory Concentrations μg/ml                                                            Ampicillin in                                           Clavulanic            presence of                                             acid                  1 μg/ml clavu-                                       sodium                lanic acid                                  Bacterial strain                                                                          salt        Ampicillin                                                                              sodium salt                                 ______________________________________                                        Escherichia coli                                                                          31        1.8         <0.4                                        NCTC 10481                                                                    Escherichia coli                                                                          62        >500        125                                         B 11                                                                          Klebsiella  31        125         <0.4                                        aerogenes A                                                                   Klebsiella sp 62                                                                          31        125         <0.4                                        Enterobacter                                                                              62        250         62                                          cloacae                                                                       Serratia    125       >500        62                                          marcescens                                                                    Staphylococcus                                                                            15        500         <0.4                                        aureus (Russell)                                                              Staphylococcus                                                                            62        250         7.5                                         aureus                                                                        ______________________________________                                    

EXAMPLE 45 Antibacterial synergism between cephaloridine and clavulanicacid sodium salt

The minimum inhibitory concentration of cephaloridine, clavulanic acidsodium salt and cephaloridine in the presence of 5 μg/ml clavulanic acidsodium salt were determined by the method described in Example 42. Theresults in Table 6 show that synergism can be obtained betweenclavulanic acid sodium salt and cephaloridine particularly for theβ-lactamase producing strain of Staphylococcus aureus (Russell).

                  TABLE 6                                                         ______________________________________                                        ANTIBACTERIAL SYNERGISM BETWEEN CEPHALORI-                                    DINE AND CLAVULANIC ACID SODIUM SALT                                                  Minimum Inhibitory Concentrations μg/ml.                                     Clavulanic           Cephaloridine in                                         acid                 presence of                                    Bacterial sodium               5 μg/ml clavulanic                          strain    salt      Cephaloridine                                                                            acid sodium salt                               ______________________________________                                        Proteus   >500*     62         7.5                                            mirabilis                                                                     899                                                                           Staphylococcus                                                                          15        3.1        <0.03.sup.+                                    aureus (Russell)                                                              Staphylococcus                                                                          62        15         3.7                                            aureus                                                                        ______________________________________                                         *Tailing Point                                                                .sup.+ Same value obtained when synergist added at 1 μg/ml instead of      μg/ml.                                                                

EXAMPLE 44 Antibacterial synergism between clavulanic acid sodium saltand various penicillins

The results presented in Table 7 were obtained by the method describedin Example 42.

                  TABLE 7                                                         ______________________________________                                        ANTIBACTERIAL SYNERGISM BETWEEN CLAVULANIC                                    ACID SODIUM SALT AND VARIOUS PENICILLINS                                      AGAINST STRAINS OF KLEBSIELLA AEROGENES                                       Amoxycillin    Carbenicillin*                                                                             Benzylpenicillin                                                +5             +5           +5                                                μg/ml.      μg/ml.    μg/ml.                           Strain                                                                              Alone   synergist                                                                              Alone synergist                                                                            Alone synergist                           ______________________________________                                        A     500     0.97     500   7.8    250   7.8                                 E 70  500     3.9      500   15     500   15.6                                62    250     15.6     125   7.8    250   15.6                                ______________________________________                                         *Similar results observed when carbenicillin replaced by carbenicillin        phenyl ester or ticacillin.                                              

EXAMPLE 45 Antibacterial synergism between ampicillin and esters ofclavulanic acid

The results presented in Table 8 were obtained by the method describedin Example 42

                  TABLE 8                                                         ______________________________________                                        ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN                                    AND ESTERS OF CLAVULANIC ACID AGAINST STRAINS                                 OF KLEBSIELLA AEROGENES                                                                           Ampicillin +                                                                             Ampicillin +                                                       5 μg/ml of                                                                            5 μg/ml of                                                      Methyl Ester                                                                             Benzyl Ester                                                       of         of                                                    Ampicillin   clavulanic clavulanic                                     Strain Alone        acid       acid                                           ______________________________________                                        A      500          1.9        1.9                                            E 70   500          3.9        3.9                                            62     500          3.9        3.9                                            ______________________________________                                    

Neither clavulanic acid methyl ester nor clavulanic acid benzyl esterinhibited the growth of the test organisms at a concentration of 100μg/ml.

EXAMPLE 46 Antibacterial activity of clavulanic acid ester

The method of Example 30 but using a dilution of 1/100 of overnightbroth, the MIC values in Table 9 were obtained for certain esters ofclavulanic acid against a number of organisms:

                  TABLE 9                                                         ______________________________________                                        ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID                                     ESTERS                                                                               MIC of Ester of Clavulanic Acid                                                                   MIC*                                                                        Piva-         of                                                              loyloxy-                                                                              Phtha-                                                                              clavulanic                                      Benzyl  Nonyl   methyl  lidyl acid sodium                            Organism ester   ester   ester   ester salt                                   ______________________________________                                        Bacillus 250     31      62      125   62                                     subtilis A                                                                    Staph. aureus                                                                           62     31      31      31    15                                     Oxford                                                                        Staph. aureus                                                                          125     31      62      15    15                                     Russell                                                                       Escherichia                                                                            125     250     125     125   125                                    coli 10418                                                                    ______________________________________                                         *The MIC of clavulanic acid sodium salt is included for comparison; the       high MIC values (if compared to those of Example 30) are due to the heavy     inocula used.                                                            

EXAMPLE 47 Extraction of clavulanic acid using liquid ion Exchange resin

Culture filtrate (200 ml, obtained in a similar manner to Example 3 butusing a medium containing 0.1% v/v KH₂ PO₄ instead of 0.01% FeSO₄.7H₂ O)was extracted with Amberlite* LA2⁺ (Cl⁻ form, 15% v/v in methylisobutylketone, 66 ml) for 30 minutes at 5° C.

The phases were separated by centrifugation (1660 g, 20 minutes). Thesolvent phase (60 ml) was recovered by pipette and divided into fourequal portions. Each portion was extracted by stirring at 5° C. for 20minutes with 1/4 volume (3.75 ml) aqueous extractant as indicated in thetable below. The resulting mixture was centrifuged (1660 g, 15 minutes).3.6 ml. aqueous phase was recovered from each extraction.

    ______________________________________                                                             Clavulanic acid                                                      Volume   concentration                                                                              Clavulanic                                  Sample      (ml)     (μg ml.sup.-1)                                                                          acid (mg)                                   ______________________________________                                        clarifield brew                                                                           200      128          25.4                                        extracted brew                                                                            200       15          3.0                                         M NaCl extract                                                                            3.6      305          1.1                                         2M NaCl extract                                                                           3.6      598          2.5                                         M NaNO.sub.3 extract                                                                      3.6      638          2.3                                         2M NaNO.sub.3 extract                                                                     3.6      758          2.73                                        ______________________________________                                    

The result obtained with 2M NANO₃ represents a recovery of 43% fromclarified brew.

EXAMPLE 48 Extraction of clavulanic acid using liquid ion Exchange resin

Clarified brew (47 liters, obtained as in Example 12) was extracted withAmberlite LA2 (acetate form, 15v/v in methylisobutyl ketone, 12.5liters) by stirring for 1 hour at 17° C. After adding octan-1-ol (500ml) the phases were separated in a continuous flow centrifuge yielding9.2 liters solvent phase, which was then stirred at 5° C. for 11/2 hourswith molar sodium nitrate (2.3 liters). The mixture was separated bycontinuous flow centrifugation yielding 2.4 liters aqueous phase(including water used for displacement purposes). Aqueous phase pH(initially 8.0) was adjusted to 7.0 with concentrated hydrochloric acid.

    ______________________________________                                                              clavulanic acid                                                                           clavulanic                                             Volume     concentration                                                                             acid                                        Sample     (1)        (μg ml.sup.-1)                                                                         (mg)                                        ______________________________________                                        clarified brew                                                                           47          146        6862                                        extracted brew                                                                           47          19          893                                        M NaNO.sub.3 extract                                                                     2.4        1638        3931                                        ______________________________________                                    

Extraction efficiency from clarified brew to sodium nitrate extract is57%.

What we claim is:
 1. A method of effecting β-lactamase inhibition in ahuman or animal in need thereof arising from a β-lactamase producingbacteria which comprises administering to said human or animal aβ-lactamase inhibitory amount of clavulanic acid or a pharmaceuticallyacceptable salt thereof.
 2. A method according to claim 1 whereinclavulanic acid is administered.
 3. A method according to claim 1wherein a pharmaceutically acceptable salt of clavulanic acid isadministered.
 4. A method according to claim 3 wherein the salt is thesodium salt.
 5. A method according to claim 3 wherein the salt is thepotassium salt.
 6. A method according to claim 3 wherein the salt is thecalcium salt.
 7. A method according to claim 3 wherein the salt is themagnesium salt.
 8. A method according to claim 3 wherein the salt is analuminum salt.
 9. A method according to claim 3 wherein the salt is theammonium salt.
 10. A method according to claim 3 wherein the salt is thetrimethylammonium salt.
 11. A method according to claim 3 wherein thesalt is the benzathine salt.
 12. A method according to claim 1 whereinthe administration is oral.
 13. A method according to claim 1 whereinthe administration is parenteral.
 14. A method according to claim 1wherein the administration is by injection.
 15. A method according toclaim 1 wherein the administration is by infusion.